Genetic determinants of micronucleus formation in vivo.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

Comparison of surgical techniques for optimal lead placement in sacral neuromodulation: a cadaver study.

BACKGROUND: Sacral neuromodulation (SNM) is a common treatment for patients with urinary and faecal incontinence. A close contact of the tined lead electrode with the targeted nerve is likely to improve functional outcome. The aim of this study was to compare the position of the SNM lead in relation to the sacral nerve by comparing different implantation techniques. METHODS: This cadaver study was conducted at the Division of Anatomy of Vienna's Medical University in October 2020. We dissected 10 cadavers after bilateral SNM lead implantation (n = 20), using two different standardized implantation techniques. The cadavers were categorized as group A (n = 10), representing the conventional guided implantation group and group B (n = 10), where SNM implantation was conducted with the novel fluoroscopy-guided "H"-technique. The primary goal was to assess the distance between the sacral nerve and the lead placement. RESULTS: The electrodes were inserted at a median angle of 58.5° (46-65°) in group A and 60° (50-65°) in group B, without reaching statistical significance. In 8 cadavers, the lead entered the S3 foramen successfully. The median distance of the lead to the nerve did not show a significant difference between both groups (E0: Group A: 0.0 mm vs. Group B: 0.0 mm, p = 0.969; E1: Group A: 0.0 mm vs. Group B: 0.5 mm p = 0.754; E2: Group A: 2.5 mm vs. Group B: 2.5 mm p = 1.000; E3: Group A: 3.5 mm vs. Group B: 4.0 mm p = 0.675). In 2 cases (20%) of the conventional group A, the lead was misplaced and located at the gluteal muscle. Perforation of the presacral fascia was observed in one lead placement in group A and in two placements in group B. CONCLUSIONS: Both standardized implantation techniques may ensure close electrode proximity to the targeted nerve. Misplacement of the electrode was more often observed with the conventional implantation technique.

Ultrasound for the detection of the pyramidal lobe of the thyroid gland.

PURPOSE: The pyramidal lobe (PL) is an ancillary lobe of the thyroid gland that can be affected by the same pathologies as the rest of the gland. We aimed to assess the diagnostic performance of high-resolution sonography in the detection of the PL with verification by dissection and histological examination. METHODS: In a prospective, cross-sectional mono-center study, 50 fresh, non-embalmed cadavers were included. Blinded ultrasound examination was performed to detect the PL by two investigators of different experience levels. If the PL was detected with ultrasound, dissection was performed to expose the PL and obtain a tissue sample. When no PL was detected with ultrasound, a tissue block of the anterior cervical region was excised. An endocrine pathologist microscopically examined all tissue samples and tissue blocks for the presence of thyroid parenchyma. RESULTS: The prevalence of the PL was 80% [40/50; 95% CI (68.9%; 91.1%)]. Diagnostic performance for both examiners was: sensitivity (85.0%; 42.5%), specificity (50.0%; 60.0%), positive predictive value (87.2%; 81.0%), negative predictive value (45.5%; 21.0%) and accuracy (78.0%; 46.0%). Regression analysis demonstrated that neither thyroid parenchyma echogenicity, thyroid gland volume, age nor body size proved to be covariates in the accurate detection of a PL (p > .05). CONCLUSION: We report that high-resolution ultrasound is an adequate examination modality to detect the PL. Our findings indicate a higher prevalence than previously reported. Therefore, the PL may be regarded as a regular part of the thyroid gland. We also advocate a dedicated assessment of the PL in routine thyroid ultrasound.

Standardized fluoroscopy-guided implantation technique enables optimal electrode placement in sacral neuromodulation: a cadaver study.

BACKGROUND: Sacral neuromodulation (SNM) is an established treatment option for patients with faecal incontinence. The location of the stimulating electrode is considered to be essential for treatment success. The purpose of this study was to evaluate the position of SNM electrodes after using a standardized fluoroscopy-guided implantation technique. METHODS: For this cadaver study, SNM electrodes were implanted bilaterally in 5 lower body specimens. The lower edge of the sacroiliac joint and the medial edge of the sacral foramina were marked using fluoroscopy to draw an 'H' with the crossing points identifying S3. After electrode placement the pelvis was dissected to describe the exact position of the SNM electrodes. RESULTS: The electrodes were inserted at an angle with a median degree measure of 60° (range 50-65°) to the skin, with a median distance of 9 mm (range 0-13 mm) from the S3 marking. All electrodes entered the third sacral foramen. The median distance of the electrodes to the sacral nerve was 0 mm (range 0-3 mm) for the most proximal, 0.5 mm (range 0-5 mm) for the second, 2.25 mm (range 0-11 mm) for the third and 1.75 mm (range 0-16 mm) for the most distant electrode. There was neither a significant difference in the proximity of the electrodes to the nerve between the right and left side (proximal to distal electrode: p = 0.18, p = 0.16, p = 0.07, p = 0.07) nor between male and female cadavers (p = 0.25, p = 0.21, p = 0.66, p = 0.66). CONCLUSIONS: A standardized fluoroscopy-guided implantation technique enables a close contact between electrode and nerve. This can potentially result in an improved clinical outcome.

Neutrophil gelatinase associated lipocalin (NGAL) is elevated in type 2 diabetics with carotid artery stenosis and reduced under metformin treatment.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL), an acute phase protein released by neutrophils, has been described as biomarker of inflammatory states. Type 2 diabetes mellitus (T2DM) is characterized by increased inflammation and an elevated risk for embolization of carotid artery stenosis (CAS). We aimed to explore the role of NGAL systemically and in plaques of diabetics undergoing carotid endarterectomy. Moreover, the potential anti-inflammatory effect of metformin on NGAL was addressed in diabetics. METHODS: Serum NGAL and matrix metalloproteinase (MMP)-9/NGAL levels were measured in 136 patients (67 with T2DM vs. 69 non-diabetics) by specific ELISA. Endarterectomy samples were graded histologically according to the American Heart Association´s classification. NGAL mRNA expression was detected using RealTime-PCR in carotid endarterectomy specimens. RESULTS: Serum NGAL [median 107.4 ng/ml (quartiles: 75.2-145.0) vs. 64.4 (50.4 -81.3), p < 0.0001] and MMP-9/NGAL [41.5 ng/ml (20.8-63.9) vs. 27.6 (16.0-42.4), p = 0.017] were significantly elevated in diabetics compared to non-diabetics, as were leukocytes, neutrophils, C-reactive protein and fibrinogen (all p < 0.05). In patients with symptomatic and asymptomatic CAS diabetics had higher NGAL levels compared to non-diabetics [128.8 ng/ml (100.8-195.6) vs. 64.8 (48.9-82.2] and [101.6 ng/ml (70.1-125.3) vs. 63.8 (51.0-81.3), respectively, both p < 0.0001]. Presence of T2DM and type VI plaques (with surface defect, hemorrhage or thrombus) had a profound impact on NGAL levels (both p < 0.01) in multiple linear regression analysis. NGAL mRNA was detectable in 95% of analyzed carotid artery lesions of diabetics compared to 5% of non-diabetics (p < 0.0001). Accordingly, cerebral embolization was more frequent in diabetics (52.2% vs. 29%, p = 0.006). Metformin treatment was associated with decreased NGAL [60.7 ng/ml (51.9-69.2) vs. 121.7 (103.7-169.9), p < 0.0001] and MMP-9/NGAL [20.8 ng/ml (12.1-26.5) vs. 53.7 (27.4-73.4), p = 0.007] in diabetics and reduced leukocyte infiltration in carotid lesions of diabetics. CONCLUSIONS: Higher NGAL levels in serum and plaques are associated with T2DM in patients with CAS. Metformin significantly reduced the inflammatory burden including NGAL in diabetics. Early treatment of these patients may be recommended, as elevated NGAL levels were linked with vulnerable plaques prone for embolization.

The dermal arteries of the human thumb pad.

The arteries of the skin have been postulated to form a profound plexus at the dermal/hypodermal junction and a superficial plexus in the papillary dermis. Our article aims to rebut this concept and to provide an alternative description of the arrangement of the dermal arteries. Employing a novel technique, we produced digital volume data (volume size: 2739 × 2054 × 3000 µm(3) ; voxel size: 1.07 × 1.07 × 2 µm(3) ) from biopsies of the skin of the thumb pads of 15 body donors. Utilizing these data, we analysed the arrangement of the dermal arteries with the aid of virtual re-sectioning tools, and, in three specimens, with high-quality three-dimensional (3D) surface models. In all specimens we observed a tree-like ramification of discrete dermal arteries. The terminal branches of the arterial trees gave rise to the ascending segments of the capillary loops of the dermal papillae. None of the specimens showed a superficial arterial plexus. This suggests that the skin of the human thumb pad can be split in discrete 'arterial units'. Each unit represents the zone of the papillary dermis and epidermal/dermal junction, to which blood is supplied exclusively by the branches of a single dermal artery. The concept of dermal arterial units is in contrast to all existing descriptions of the architecture of the dermal arteries. However, whether it can be transferred to the skin of other body parts, remains to be tested. Likewise, the consequences of arterial units for understanding the mechanisms of wound healing and the appearance and genesis of skin diseases remain to be examined.

A chick embryo with a yet unclassified type of cephalothoracopagus malformation and a hypothesis for explaining its genesis.

Cephalothoracopagus embryos are conjoined twins, who share parts of their heads, necks and bodies. Our study aims at presenting a detailed morphological analysis of a cephalothoracopagus chick embryo of developmental stage 31. Because none of the existing theories can explain the genesis of the phenotype of this embryo, we also suggest a hypothesis, which explains it. Beside the cephalothoracopagus embryo, we investigated five control embryos. With the aid of the high-resolution episcopic microscopy (HREM) technique, we created digital volume data and three-dimensional (3D) computer models of the organs and arteries of the embryos. We used the 3D models for topological analysis and for measuring the diameters of the great intrathoracic arteries. The malformed embryo showed two body backs, each containing a notochord, spinal cord and dorsal aorta. The body backs continued into separated lower bodies. The embryo had a single, four-chambered heart, single respiratory tract and single upper alimentary tract. The topology of the pharyngeal arch arteries was normal, and the diameters of these arteries were similar to that of the control embryos. We classified the embryo we investigated as a yet unknown malformation and suggest a hypothesis explaining its genesis.

Ret finger protein inhibits muscle differentiation by modulating serum response factor and enhancer of polycomb1.

Skeletal myogenesis is precisely regulated by multiple transcription factors. Previously, we demonstrated that enhancer of polycomb 1 (Epc1) induces skeletal muscle differentiation by potentiating serum response factor (SRF)-dependent muscle gene activation. Here, we report that an interacting partner of Epc1, ret finger protein (RFP), blocks skeletal muscle differentiation. Our findings show that RFP was highly expressed in skeletal muscles and was downregulated during myoblast differentiation. Forced expression of RFP delayed myoblast differentiation, whereas knockdown enhanced it. Epc1-induced enhancements of SRF-dependent multinucleation, transactivation of the skeletal α-actin promoter, binding of SRF to the serum response element, and muscle-specific gene induction were blocked by RFP. RFP interfered with the physical interaction between Epc1 and SRF. Muscles from rfp knockout mice (Rfp(-/-)) mice were bigger than those from wild-type mice, and the expression of SRF-dependent muscle-specific genes was upregulated. Myotube formation and myoblast differentiation were enhanced in Rfp(-/-) mice. Taken together, our findings highlight RFP as a novel regulator of muscle differentiation that acts by modulating the expression of SRF-dependent skeletal muscle-specific genes.

Measurements of the diameters of the great arteries and semi-lunar valves of chick and mouse embryos.

The great arteries of embryos are small channels of a complex three-dimensional arrangement. Measurements of their diameters, as required for understanding cardiovascular morphogenesis and the genesis of malformations, cannot be performed in two-dimensional histological sections. We present and evaluate a quick and simple method for performing highly significant and objective measurements of the diameters of blood vessels in vertebrate embryos and used this method for providing statistics of the diameter of the semi-lunar valves and the lumina of the great arteries of early chick and mouse foetus. We employed the high-resolution episcopic microscopy technique for generating volume data and three-dimensional computer models of the arterial trees of 30 chick embryos (Hamburger Hamilton stage 34), 30 mouse embryos of the OF1 strain harvested on 14.5 dpc, 30 embryos of the OF1 strain harvested on 15.5 dpc and 28 mouse embryos of the PARKES strain harvested on 14.5 dpc. The three-dimensional models (voxel size 2 mum x 2 mum x 2 mum and 3 mum x 3 mum x 3 mum) were used for defining virtual resection planes perpendicular to the longitudinal axis of the blood vessels at comparable positions. In these planes, we measured the lumen areas and the lumen perimeters. We also calculated the lumen diameter and the true lumen area from the perimeter and present statistical analysis. Finally, we evaluate and discuss the reliability and reproducibility of our method and present all measurements in a form that minimizes the influence of specimen size variation, specimen processing and data generation methods.

[Morphology and functional anatomy of the growing thorax]. / Morphologie und funktionelle Anatomie des wachsenden Thorax.

Morphogenesis of most thoracic organs and structures is not finished with birth but perpetuates in postnatal life. The postnatal growth is partly associated with enormous changes in structure, morphology, and function. Our study presents an overview of morphological, topological and functional peculiarities of thoracic anatomy during infancy, childhood and adolescence. It focuses on the development of the mammary gland, osseous structures of the chest wall, thymus, heart, and lungs. Most of the presented data are based on post-mortem studies. Measurements and numerical data are mainly included for illustration of growth-associated changes.

The morphology of heart development in Xenopus laevis.

We have used serial histological sections to document heart formation in Xenopus laevis, from the formation of a linear heart tube to the appearance of morphologically distinct atrial and ventricular chambers. 3D reconstruction techniques have been used to derive accurate models from digital images, revealing the morphological changes that accompany heart differentiation. To demonstrate the utility of this approach in analysing cardiac gene expression, we have reexamined the distribution of Hand1 transcripts in the linear and looped heart tube. Our results demonstrate that prior to looping, an initial asymmetric, left-sided pattern is replaced by more symmetrical localisation of transcripts to the ventral portion of the myocardium. After the onset of looping, Hand1 expression is restricted to the ventral ventricular myocardium and extends along the entire length of the single ventricle.

Compartments of the adult parasellar region.

In the infant the region lateral to the sella turcica which is traditionally termed the 'cavernous sinus' is composed of 3 individual compartments. The aim of this study was to demonstrate a similar compartmentation of the adult cavernous sinus and to identify and quantify the adipose bodies which are located within the compartments. The region of the cavernous sinus in 136 adults was analysed by microdissection and histology. We demonstrate that in 66% of the cavernous sinus in adults is composed of at least 2 compartments and in 22% it is made up of 3. Assimilating the renaming in infants we termed the compartments 'pterygopalatine', 'orbital' and 'lateral sellar'. The pterygopalatine and orbital compartments are connected with extracranial tissue spaces via the superior orbital fissure and contain characteristic adipose bodies. Exact topographic descriptions and measurements of the compartments and their adipose bodies are provided. Our study clearly defines compartmentation of the adult parasellar space in most individuals and thus changes the anatomical view of this space. The direct connection of 2 of these compartments with extracranial tissue spaces and the measurements of their adipose bodies are of interest for surgeons and neuroradiologists.

Intimal hyperplasia of the infant parasellar carotid artery: a potential developmental factor in atherosclerosis and SIDS.

Intimal cushions that project into the lumen of arteries are precursors of atherosclerotic plaque formation. The "carotid siphon, " although frequently affected by atherosclerosis, was never analyzed for the occurrence of neonatal intimal hyperplasia. This study provides a topographic and morphometric analysis of intimal cushions in the parasellar internal carotid artery (pICA) of the human infant. A total of 35 specimens were studied in detail, using both standard histological techniques and a new method of computer-aided 3D reconstruction. Intimal hyperplasia occurred at 3 characteristic locations of the pICA: (1) the convex side of the posterior knee (C5 cushion), (2) the bottom of the horizontal segment (C4 cushion), and (3) the concave side of the anterior knee (C3 cushion). The extension of the cushions and the degrees to which they occluded the vessel lumens were measured. The complex shape of the pICA required 3D computer models for exact topographical descriptions and precise measurements. Our results suggest that the occurrence and degree of intimal hyperplasia are related to shape changes of the pICA during postnatal development. We predict that individuals who retain the relatively straight course of the fetal pICA throughout their lives are less prone to develop atherosclerotic lesions at this portion of the carotid artery. A possible contribution of neonatal intimal cushions to the origin of sudden infant death syndrome is discussed.

The parasellar region of human infants: cavernous sinus topography and surgical approaches.

OBJECT: In this study the authors analyze the peculiarities of the parasellar anatomy and the topography of surgical approaches to the parasellar region (PSR) in human infants. METHODS: Forty-nine specimens of the PSR obtained at autopsy were studied using microdissection and histological analysis. Important distances between anatomical landmarks were measured with the aid of a dissecting microscope. One serially sectioned specimen was three-dimensionally reconstructed and analyzed on the computer screen by using the authors' new episcopic reconstruction technique. CONCLUSIONS: The anatomy of the infant PSR differs distinctly from that of the adult. The parasellar portion of the internal carotid artery (ICA) does not form a siphon, but takes a straight course, and the venous pathways as well as the cranial and sympathetic nerves have different topographical relationships. Analyses of surgical approaches demonstrate that, in young children, the anterolateral approach can be used to reach the pterygopalatine compartment, the superior ophthalmic vein, and those pathological processes that extend from the orbit into the PSR. The approach via Parkinson's triangle can be used in 45% of cases to access the pathological processes that occur in the voluminous space above and behind the posterior flexure of the parasellar ICA. Taking this route, sympathetic nerve fibers passing through the PSR are not at risk, but some arterial branches that run within the lateral wall of the sinus can complicate this approach. This study presents a guideline that can assist radiologists and neurosurgeons in the planning and performance of interventions within the PSR of neonates and young children.

A new episcopic method for rapid 3-D reconstruction: applications in anatomy and embryology.

The topographic relations of complex structures and the morphogenesis of organ systems can only be fully understood in their three-dimensional context. Three-dimensional (3-D) reconstruction of physically sectioned specimens has become an indispensable tool in modern anatomical and embryological research. Teaching also makes increasingly use of 3-D representations, in particular in the case of embryonic systems that undergo complicated transformations of form and shape. At present no cheap and simple technique is available that generates accurate 3-D models of sectioned objects. In this study we describe a novel technique that rapidly provides faithful 3-D models of sectioned specimens. The images are captured directly from the cutting surface of the embedding block after each sectioning and "on block" staining step. Automatic image processing generates a stack of binary images of the specimen contour. Binary images of internal structures are obtained both by automatic segmentation and manual tracing. Since these image series are inherently aligned, they can be reconstructed three-dimensionally without time-consuming alignment procedures. The quality and the flexibility of the method are demonstrated by reconstructing three kinds of specimens of different histological composition and staining contrast: a 4 mm mouse embryo together with several of its inner organs, a cavernous sinus region of a human infant, and a segment of a human carotid artery. Very short processing times and the faithful representation of complex structural arrangements recommend this technique for routine use in morphological research and for creating embryologic teaching models or 3-D embryonic staging series.

External marker-based automatic congruencing: a new method of 3D reconstruction from serial sections.

BACKGROUND: Computer-based three-dimensional (3D) visualizations reconstructed from sectional images represent a valuable tool in biomedical research and medical diagnosis. Particularly with those imaging techniques that provide virtual sections, such as CT, MRI, and CLSM, 3D reconstructions have become routine. Reconstructions from physical sections, such as those used in histological preparations, have not experienced an equivalent breakthrough, due to inherent shortcomings in sectional preparation that impede automated image-processing and reconstruction. The increased use of molecular techniques in morphological research, however, generates an overwhelming amount of 3D molecular information, stored within series of physical sections. This valuable information can be fully appreciated and interpreted only through an adequate method of 3D visualization. METHODS AND RESULTS: In this paper we present a new method for a reliable and largely automated 3D reconstruction from physically sectioned material. The 'EMAC' concept (External Marker-based Automatic Congruencing) successfully approaches the three major obstacles to automated 3D reconstruction from serial physical sections: misalignment, distortion, and staining variation. It utilizes the objectivity of external markers for realignment of the sectional images and for geometric correction of distortion. A self-adapting dynamic thresholding technique compensates for artifactual staining variation and automatically selects the desired object contours. CONCLUSIONS: Implemented on a low-cost hardware platform, EMAC provides a fast and efficient tool that largely facilitates the use of computer-based 3D visualization for the analysis of complex structural, molecular, and genetic information in morphological research. Due to its conceptual versatility, EMAC can be easily adapted for a broad range of tasks, including all modern molecular-staining techniques, such as immunohistochemistry and in situ hybridization.

Anatomical compartments of the parasellar region: adipose tissue bodies represent intracranial continuations of extracranial spaces.

The cavernous sinus is traditionally described as a single anatomical compartment that contains cranial nerves, blood vessels, and connective tissue. A detailed analysis of 45 infant and 4 fetal parasellar regions shows that this view must be modified. The spatial arrangement, the topographic relations, and the expansion of the adipose and connective tissue spaces were analysed and reconstructed 3-dimensionally on a computer. It is shown that 3 different anatomical compartments, which are strictly demarcated by connective tissue, compose the parasellar region of infants. Two represent intracranial continuations of extracranial tissue spaces. The 3rd compartment corresponds to the so-called 'cavernous sinus' of the adult. Each of the 3 compartments contains characteristic adipose tissue bodies. Because the cavernous sinus represents only one compartment of the area, we propose to use the term 'parasellar region' to designate the entire anatomical region on either side of the sella turcica.

The sympathetic nerves of the parasellar region: pathways to the orbit and the brain.

Sympathetic nerves innervate targets in the orbit and the brain. They issue from the superior cervical ganglion and reach the parasellar region via the internal carotid nerve. Information on their further parasellar course and distribution is scant and contradictory. In this study the parasellar sympathetic pathways of 30 human infants and 6 human fetuses were investigated by microdissection and histologically. A common parasellar sympathetic trunk, which reunites all the nerve fibers emanating from the lateral and medial internal carotid plexus, is described as well as its further divisions. It was found that the posterior knee of the infant carotid siphon is free of large sympathetic nerve bundles. In addition a ganglion is described, which is situated in the parasellar adipose body. It is reached by nerve fibers coming from the parasellar sympathetic pathways. Fibers that issue from this ganglion join the periorbita and the orbital muscle of Müller. These anatomical facts are of immediate importance for preventing nerve damage during cavernous sinus surgery. Furthermore, the study improves the anatomical knowledge of the parasellar region and suggests a new concept for the innervation of the orbital muscle.

[3-dimensional reconstruction of histological serial sections using a computer]. / Dreidimensionale Rekonstruktion histologischer Schnittserien am Computer.

This paper reports the development of an objective and time-saving method for the three-dimensional reconstruction of sectioned morphological specimens on the computer. The development of a fiducial marker technique, the design of program extensions for the automatic thresholding and realignment of images, and a technique for the transfer of marker-derived reference points into images of higher magnification are described. The method is based on the use of resin as embedding material and drill holes as fiducials. Images are digitized by a video camera, and NIH-Image is used for image processing. Special macros were written for the automation of a number of operations, such as object and marker segmentation, image reorientation, image scaling, dirt removal, and marker transfer. The so-treated image series were transferred to a workstation and were reconstructed using "Image Volumes". The reconstructs obtained from this largely automated technique exhibit a perfect alignment of the sections. The 3-D representations of the sectioned objects have a much higher degree of objectivity than those obtained by manual image orientation or by semiautomated techniques that do not use fiducials.