Extraordinary branching pattern of the aortic arch.

A CT-scan of a 75-year-old patient showed an aneurysm of the descending aorta with a maximum diameter of 4.8 cm involving the left subclavian artery. Due to the fact that the patient had several comorbidities including a severe chronic obstructive pulmonary disease he was treated only conservatively. However, there were several interesting findings on the CT-scan: the branching pattern of the aortic arch revealed a left carotid artery arising as first side branch of the distal part of the ascending aorta. This vessel crosses the midline right in front of the trachea. Apart from that the patient did not have a brachiocephalic trunk: the right carotid artery arose as the first branch from the aortic arch and crossed the right subclavian artery anteriorly. Furthermore, the left subclavian seemed to arise from the descending aorta and not from the aortic arch.

VEGF and its role in the early development of the long bone epiphysis.

In long bones of murine species, undisturbed development of the epiphysis depends on the generation of vascularized cartilage canals shortly after birth. Despite its importance, it is still under discussion how this event is exactly regulated. It was suggested previously that, following increased hypoxia in the epiphyseal core, angiogenic factors are expressed and hence stimulate the ingrowth of the vascularized canals. In the present study, we tested this model and examined the spatio-temporal distribution of two angiogenic molecules during early development in mice. In addition, we investigated the onset of cartilage hypertrophy and mineralization. Our results provide evidence that the vascular endothelial growth factor is expressed in the epiphyseal resting cartilage prior to the moment of canal formation and is continuously expressed until the establishment of a large secondary ossification centre. Interestingly, we found no expression of secretoneurin before the establishment of the canals although this factor attracts blood vessels under hypoxic conditions. Epiphyseal development further involves maturation of the resting chondrocytes into hypertrophic ones, associated with the mineralization of the cartilage matrix and eventual death of the latter cells. Our results suggest that vascular endothelial growth factor is the critical molecule for the generation of the epiphyseal vascular network in mice long bones. Secretoneurin, however, does not appear to be a player in this event. Hypertrophic chondrocytes undergo cell death by a mechanism interpreted as chondroptosis.

Computer-assisted surgery in the edentulous jaw based on 3 fixed intraoral reference points.

PURPOSE: In computer-assisted implantation surgery, the transfer of prosthodontic-guided planning to the operative site is usually based on a registration template. The precise repositioning of the registration template is crucial for high accuracy and is compromised in edentulous jaws. The purpose was to determine the in vitro registration and targeting accuracy for surgical navigation in the edentulous jaw based on 3 fixed intraoral reference points. MATERIALS AND METHODS: Edentulous maxilla and mandible cadaver specimens were provided with 3 fixed reference-point screws. A resin template with matrices for the fixed reference-point screws was produced and connected to a Vogele-Bale-Hohner registration mouthpiece and external registration frame with a snap-lock system. Surgical implants were planned on computed tomographic data and the corresponding dental stone casts were drilled under guidance of an optical navigation system. For evaluation of the registration accuracy, fiducial registration error was recorded and application accuracy was evaluated by fusion of postsurgical computed tomographic scans of the drilled dental stone casts with the presurgical planning computed tomogram. RESULTS: In 9 maxillas and 5 mandibles, 14 registrations and 104 stone cast drillings were performed. The mean fiducial registration error was 0.49 +/- 0.14 mm (0.37 to 0.9 mm). The mean total error at the tip of the borehole was 0.88 +/- 0.65 mm (0.0 to 4.24 mm). The mean lateral errors were 0.51 +/- 0.49 mm (0.0 to 2.80 mm) at the base and 0.46 +/- 0.34 mm (0.0 to 1.5 mm) at the tip of the borehole, respectively. The mean angular error was 0.83 +/- 0.60 degrees (0.0 to 2.5 degrees ). CONCLUSION: Three fixed intraoral reference points successfully support a registration mouthpiece and provide in vitro registration and targeting accuracy that is comparable to tooth-supported registration templates or bone marker registration.

Localization of tartrate-resistant acid phosphatase (TRAP), membrane type-1 matrix metalloproteinases (MT1-MMP) and macrophages during early endochondral bone formation.

Endochondral bone formation, the process by which most parts of our skeleton evolve, leads to the establishment of the diaphyseal primary (POC) and epiphyseal secondary ossification centre (SOC) in long bones. An essential event for the development of the SOC is the early generation of vascularized cartilage canals that requires the proteolytic cleavage of the cartilaginous matrix. This in turn will allow the canals to grow into the epiphysis. In the present study we therefore initially investigated which enzymes and types of cells are involved in this process. We have chosen the mouse as an animal model and focused our studies on the distal part of the femur during early stages after birth. The formation of the cartilage canals was promoted by tartrate-resistant acid phosphatase (TRAP) and membrane type-1 matrix metalloproteinases (MT1-MMP). In addition, macrophages and cells containing numerous lysosomes contributed to the establishment of the canals and enabled their further advancement into the epiphysis. As development continued, the SOC was formed, and in mice aged 10 days a distinct layer of type I collagen (= osteoid) was laid down onto the cartilage scaffold. The events leading to the establishment of the SOC were compared with those of the POC. Basically these processes were quite similar, and in both ossification centers, TRAP-positive chondroclasts resorbed the cartilage matrix. However, occasionally co-expression of TRAP and MT1-MMP was noted in a small subpopulation of this cell type. Furthermore, numerous osteoblasts expressed MT1-MMP from the start of endochondral ossification, whereas others did not. In osteocytogenesis, MT1-MMP has been shown to be critical for the establishment of the cytoplasmic processes mediating the communication between osteocytes and bone-lining cells. Considering the well-known fact that not all osteoblasts transform into osteocytes, and in accordance with the present data, we suggest that MT1-MMP is needed at the very beginning of osteocytogenesis and may additionally determine whether an osteoblast further differentiates into an osteocyte.

Structure, formation and role of cartilage canals in the developing bone.

In the long bones, endochondral bone formation proceeds via the development of a diaphyseal primary ossification centre (POC) and an epiphyseal secondary ossification centre (SOC). The growth plate, the essential structure for longitudinal bone growth, is located between these two sites of ossification. Basically, endochondral bone development depends upon neovascularization, and the early generation of vascularized cartilage canals is an initial event, clearly preceding the formation of the SOC. These canals form a discrete network within the cartilaginous epiphysis giving rise to the formation of the marrow space followed by the establishment of the SOC. These processes require excavation of the provisional cartilaginous matrix which is eventually replaced by permanent bone matrix. In this review, we discuss the formation of the cartilage canals and the importance of their cells in the ossification process. Special attention is paid to the enzymes required in disintegration of the cartilaginous matrix which, in turn, will allow for the invasion of new vessels. Furthermore, we show that the mesenchymal cells of the cartilage canals express bone-relevant proteins and transform into osteocytes. We conclude that the canals are essential for normal epiphyseal bone development, the establishment of the growth plate and ultimately longitudinal growth of the bones.

Microscopy of the Drosophila facet eye: vademecum for standardized fixation, embedding, and sectioning.

We describe here a standardized method for histological processing of the Drosophila compound eye. Primary fixation with 2.5% glutaraldehyde, obligatorily supplemented with 0.1% household detergent regularly yielded the best structural preservation, as compared with that of other, more complicated fixation protocols tested. Notably, it proved indispensable not only to cut off the fly's head to facilitate the penetration of the reagents but also to open the chitinous head capsule. For this, we locally pierced the cuticle between the eyes, leaving the head structurally almost intact, a prerequisite for precisely aligning the head for microtomy. We developed a two-step re-embedding procedure allowing for exact and reproducible orientation of the fly heads. Thus, highly comparable series of cross sections through a representative number of ommatidia were obtained. The feasibility of our embedding and sectioning approach is finally demonstrated by three-dimensional reconstructions of the middle segments of the R1, R7, and R8 photoreceptor cells. We present reconstructions from structurally modified ommatidia, as seen after RNAi-mediated depletion of the endosomal adaptor protein p14, and from normal ommatidia corresponding to the wildtype.

Is the bare spot a consistent landmark for shoulder arthroscopy? A study of 20 embalmed glenoids with 3-dimensional computed tomographic reconstruction.

PURPOSE: Our aim was to test a published methodology for arthroscopically quantifying glenoid bone loss for its dependability in a cadaver-based anatomic study with 3-dimensional (3-D) computed tomographic (CT) reconstructions of 20 embalmed glenoids. METHODS: Manual macroscopic measurements were made in a standardized fashion. In addition, we marked the center of the visible bare spot in 20 embalmed glenoids with a titanium pin. The shoulder joints were carefully selected for intact rotator cuff, missing capsule-labral deficiency, and absence of severe cartilage degeneration. 3-D reconstructed CT scans were evaluated for consistency of the bare spot. RESULTS: The mean distance from the bare spot to the anterior margin measured manually was 10.9 mm (CT-based, 13.9 mm), to the posterior margin 13.7 mm (CT-based, 16.4 mm), and to the inferior margin 9.7 mm (CT-based, 15.1 mm). Distances were significantly different. CONCLUSIONS: The bare spot did not prove its consistency in 20 carefully selected specimens. Therefore, we conclude that this methodology is not a reliable way to intraoperatively determine bony glenoid deficiency. CLINICAL RELEVANCE: We recommend preoperative bilateral CT scans for evaluation of bony glenoid deficiency for exact quantification of the bone loss.

Cartilage canals in the chicken embryo are involved in the process of endochondral bone formation within the epiphyseal growth plate.

A detailed study of so-called communicating cartilage canals, which penetrate deeply up into the lower hypertrophic zone of the epiphyseal growth plate in the embryonic chicken femur (E20), was carried out with the aim to clarify whether or not these canals are involved in the bone-forming process. In addition, we examined the manner in which cartilage canals are formed and compare the present data with our previous data. The canals were investigated by means of light microscopy, electron microscopy, immunohistochemistry (VEGF, VEGFR2/Flk1, type I collagen), and 3D reconstruction. Some communicating canals deeply penetrate into the upper hypertrophic zone where they terminate, showing electron-dense cells at their end. Subcellular characteristics of these cells are hardly detectable and we suppose that they undergo cell death. Other canals pass down deeper into the lower hypertrophic zone. The upper segment of these canals is composed of capillaries, mesenchymal cells, and macrophage-like cells. Precursors of osteoblasts are adjacent to the canals. The lower segment of communicating canals is composed of bone matrix or osteoid, which contains type I collagen fibrils and cells having the typical subcellular features of osteoblasts. No vessels are found in these segments. Immunohistochemistry shows that the matrix of the canals labels positively for type I collagen. In addition, staining with sirius red demonstrates that bone matrix is formed in these parts. We assume that the osteoblast-like cells of the lower segments of communicating canals originate either from mesenchymal cells or even from hypertrophic chondrocytes. Our immunohistochemical data also reveal that vascular endothelial growth factor (VEGF) and the corresponding receptor VEGFR2/Flk1 (VEGF receptor 2/Flk1) are localized in cartilage canals of the reserve zone, the proliferative zone, and the hypertrophic zone. The receptor is found in the endothelial cells of the vessels. Furthermore, VEGF is present in hypertrophic chondrocytes. The results of our study suggest that cartilage canals penetrate actively into the cartilage anlage and that bone is formed in the lower segments of the communicating canals where no vessels are detectable.

Cisatracurium, but not mivacurium, inhibits survival and axonal growth of neonatal and adult rat peripheral neurons in vitro.

Cisatracurium and mivacurium are widely used neuromuscular blocking drugs. Previous reports have indicated growth-inhibitory effects of cisatracurium, but not mivacurium, on two human cell lines in vitro. These effects were ascribed to oxidative stress elicited by acrylate esters formed during cisatracurium breakdown. The aim of the present study was to investigate whether these agents would possibly interfere with the peripheral nervous system. Survival and axonal growth of rat primary neurons obtained from the superior cervical sympathetic ganglion (SCG) or from the adult dorsal root ganglion (DRG) were investigated after treatment with cisatracurium or mivacurium at concentrations from 1 to 10 microM for 24 h. Cisatracurium, but not mivacurium, significantly decreased neuronal survival in a dose-dependent manner, and axonal length was considerably reduced by cisatracurium as compared to controls. It is concluded that high concentrations of cisatracurium are potentially neurotoxic.

Bone development in the femoral epiphysis of mice: the role of cartilage canals and the fate of resting chondrocytes.

In mammals, the exact role of cartilage canals is still under discussion. Therefore, we studied their development in the distal femoral epiphysis of mice to define the importance of these canals. Various approaches were performed to examine the histological, cellular, and molecular events leading to bone formation. Cartilage canals started off as invaginations of the perichondrium at day (D) 5 after birth. At D 10, several small ossification nuclei originated around the canal branched endings. Finally, these nuclei coalesced and at D 18 a large secondary ossification centre (SOC) occupied the whole epiphysis. Cartilage canal cells expressed type I collagen, a major bone-relevant protein. During canal formation, several resting chondrocytes immediately around the canals were active caspase 3 positive but others were freed into the canal cavity and appeared to remain viable. We suggest that cartilage canal cells belong to the bone lineage and, hence, they contribute to the formation of the bony epiphysis. Several resting chondrocytes are assigned to die but others, after freeing into the canal cavity, may differentiate into osteoblasts.

Epithelial and muscular regionalization of the human developing anorectum.

In the past, interpretations of anorectal development were mainly based on analysis of serially sectioned embryos of various nonhuman species as well as some human specimens. A four-dimensional view of the developmental situation in the human has never been established nor connected to recent findings obtained from newer molecular techniques. We, therefore, investigated human embryonic and fetal pelves by means of immunohistochemistry and in situ hybridization to elucidate differentiation and interaction of epithelial and mesenchymal layers of the anorectum. To emphasize spatial as well as sequential morphological development, we produced three-dimensional reconstructions of the specimens at hand. Research conducted proved that the decisive steps of epithelial and muscular differentiation occur between the 7th and 9th week after conception. This study elucidates a biphasic epithelial "closure" in the anal canal and interactions between epithelium, smooth musculature, and skeletal musculature. Based on the results presented here, it is possible to describe the pathogenesis of two anorectal malformations: the imperforate anal membrane and the anal membrane stenosis. This study will now provide the basis for further research into developmental processes occurring before the ones examined.

Growth curves of the fetal prostate based on three-dimensional reconstructions: a correlation with gestational age and maternal testosterone levels.

OBJECTIVE: To create a nomogram of the fetal growth of the human prostate corresponding to gestational age, and to investigate the relationship between the expansive growth of the fetal prostate and the maternal testosterone surge during pregnancy. MATERIALS AND METHODS: In all, 27 fetal prostates at 11-40 weeks of gestation, and seven neonatal specimens at 1-20 weeks after birth, were analysed. Serial sections of prostates were immunostained and examined using light microscopy. After modular image acquisition the volumes were calculated using three-dimensional reconstruction. The prostate volumes were correlated with gestational age, and related to reference testosterone levels during pregnancy. RESULTS: There was exponential growth of the fetal prostate with gestational age. The increasing volume of the prostate during the fetal period corresponded with maternal testosterone levels. In the second trimester there was a significant increase in prostate volume in relation to the bladder. In infants, macroscopically there was an inverse proportion between bladder size and prostate volume. CONCLUSIONS: Starting from the second trimester there is distinct growth of the fetal prostate, obviously triggered by the maternal testosterone surge. In neonates there is an inversion of the dimensions between bladder and prostate. These results indicating exponential growth of the fetal prostate provide evidence of a gender-related transient infravesical obstruction in human fetuses.

Reevaluation of the fetal muscle development of the vesical trigone.

PURPOSE: Fetal development of the interureteral muscle, a precondition of a sufficient opposite anchoring of the ureterovesical junction, and the muscle architecture of the bladder neck was investigated using immunohistochemical analysis. MATERIALS AND METHODS: We investigated the ureterotrigonal units in 38 fetal specimens (16 females, 22 males) and 7 newborns (2 females, 5 males) at 9 to 40 weeks of gestation. Histology was based on serial consecutive sections of the bladder base. Anti-human alpha-smooth muscle actin immunostaining was used to demonstrate the time course of muscle development and arrangement. RESULTS: A much earlier developmental stage of the trigone muscle configuration during fetal life was noted than has been reported to date. The condensation of myoblasts located mainly in the dorsal wall of the trigone and at the bladder outlet was present beginning at 12 weeks of gestation. The trigone develops continuously as a single circular muscular layer corresponding to the posterior part of the vesical sphincter muscle. Muscle fibers forming the interureteral junction were demonstrable beginning at 14 weeks of gestation. CONCLUSIONS: There is a close connection between the trigonal smooth muscle layer and the vesical sphincter muscle forming the main part of the trigone by 12 weeks of gestation. Fetal development of the trigone, in particular the muscle architecture of the bladder neck, which consists of only a ring-shaped muscular layer, and the transverse oriented interureteral muscle results in a functional entity representing the anatomical basis for a competent ureterovesical junction.

The role of cartilage canals in endochondral and perichondral bone formation: are there similarities between these two processes?

We investigated the development of cartilage canals to clarify their function in the process of bone formation. Cartilage canals are tubes containing vessels that are found in the hyaline cartilage prior to the formation of a secondary ossification centre (SOC). Their exact role is still controversial and it is unclear whether they contribute to endochondral bone formation when an SOC appears. We examined the cartilage canals of the chicken femur in different developmental stages (E20, D2, 5, 7, 8, 10 and 13). To obtain a detailed picture of the cellular and molecular events within and around the canals the femur was investigated by means of three-dimensional reconstruction, light microscopy, electron microscopy, histochemistry and immunohistochemistry [vascular endothelial growth factor (VEGF), type I and II collagen]. An SOC was visible for the first time on the last embryonic day (E20). Cartilage canals were an extension of the vascularized perichondrium and its mesenchymal stem cell layers into the hyaline cartilage. The canals formed a complex network within the epiphysis and some of them penetrated into the SOC were they ended blind. The growth of the canals into the SOC was promoted by VEGF. As the development progressed the SOC increased in size and adjacent canals were incorporated into it. The canals contained chondroclasts, which opened the lacunae of hypertrophic chondrocytes, and this was followed by invasion of mesenchymal cells into the empty lacunae and formation of an osteoid layer. In older stages this layer mineralized and increased in thickness by addition of further cells. Outside the SOC cartilage canals are surrounded by osteoid, which is formed by the process of perichondral bone formation. We conclude that cartilage canals contribute to both perichondral and endochondral bone formation and that osteoblasts have the same origin in both processes.