The venous system of E14.5 mouse embryos-reference data and examples for diagnosing malformations in embryos with gene deletions.

Approximately one-third of randomly produced knockout mouse lines produce homozygous offspring, which fail to survive the perinatal period. The majority of these die around or after embryonic day (E)14.5, presumably from cardiovascular insufficiency. For diagnosing structural abnormalities underlying death and diseases and for researching gene function, the phenotype of these individuals has to be analysed. This makes the creation of reference data, which define normal anatomy and normal variations the highest priority. While such data do exist for the heart and arteries, they are still missing for the venous system. Here we provide high-quality descriptive and metric information on the normal anatomy of the venous system of E14.5 embryos. Using high-resolution digital volume data and 3D models from 206 genetically normal embryos, bred on the C57BL/6N background, we present precise descriptive and metric information of the venous system as it presents itself in each of the six developmental stages of E14.5. The resulting data shed new light on the maturation and remodelling of the venous system at transition of embryo to foetal life and provide a reference that can be used for detecting venous abnormalities in mutants. To explore this capacity, we analysed the venous phenotype of embryos from 7 knockout lines (Atp11a, Morc2a, 1700067K01Rik, B9d2, Oaz1, Celf4 and Coro1c). Careful comparisons enabled the diagnosis of not only simple malformations, such as dual inferior vena cava, but also complex and subtle abnormalities, which would have escaped diagnosis in the absence of detailed, stage-specific referenced data.

Sacral preauricular extensions and notches as parts of a 'Pelvic Pattern' may provide information on past pregnancies and parturitions.

During the analyses of several hundred prehistoric individuals from Austria, we observed that some women display a "Pelvic Pattern" at the innominate bones and the sacrum, i.e. specific combinations of pronounced expressions of pelvic features. We recorded classic pelvic features (dorsal pubic pitting, preauricular sulcus, extended pubic tubercle) as well as new ones (SPE: sacral preauricular extension, a ventrally pointing flat bone formation at the ventrosuperior margin of the ala ossis sacri; SPN: sacral preauricular notch, a loss of convexity at the same location; CF: corresponding facets at the ilium), and some less well-known features, i.e. the margo auricularis groove, ventral pubic exostoses and lesions. To quantify the assessed features, we developed a specific formula to calculate the 'Pelvic Pattern Index' (PPI). As pregnancies and/or parturitions are suspected to contribute to or be at least partly causative of the occurrence of pelvic features, we analyzed 48 well-preserved female individuals and 15 males from identified skeletal collections with obstetric information in Geneva and London. In these collections, we found a pelvic pattern of at least four out of ten distinctly expressed pelvic features only in multiparous females, but not in nulli- or primiparous females or in males. This pattern was found in 40.6% of the multiparous females and 29.2% of all females from the identified collections, compared to 56.1% of well-preserved prehistoric females with unknown parity status from Austria (n = 41). The mean PPI of the multiparae from the identified collections is 0.25, compared to a mean PPI of 0.19 for all women from the identified collections, and 0.28 for the prehistoric female individuals. We conclude from this that a high PPI (≥ 0.30), especially in cases where SPE or SPN are present, can give insights into past motherhood.

Laparoscopic Sacral Mesh Fixation for Ventral Rectopexy: Clinical Implications From a Cadaver Study.

BACKGROUND: Correct tack placement at the sacral promontory for mesh fixation in ventral mesh rectopexy is crucial to avoid bleeding, nerve dysfunction, and spondylodiscitis. OBJECTIVE: The present cadaver study was designed to assess the true location of tacks after mesh fixation during laparoscopic ventral mesh rectopexy in relation to vascular and nerve structures and bony landmarks. DESIGN: This was an interventional cadaver study. SETTING: This study was conducted after laparoscopic mesh fixation detailed pelvic dissection was performed following a standardized protocol. In addition, 64-row multidetector computed tomography was conducted to further define lumbosacral anatomy and tack positioning. PATIENTS: Eighteen fresh cadavers (10 female, 8 male) were included in this study. MAIN OUTCOME MEASURES: True tack position and vascular and neuronal involvement served as outcome measures. RESULTS: A total of 52 tacks were deployed (median 3, range 2-3 tacks). Median tack distance to the midsacral promontory was 16.1 mm (0.0-54.2). Only a total of 22 tacks (42.3%) were found on the right surface of the S1 vertebra, correlating with the planned deployment area. In 7 cadavers (38.8%), all tacks were deployed on the planned deployment area. The median distance to the major vessels was 10.5 mm (0.0-35.0), which was the internal iliac artery in half of the cases. Median distance of tacks to the right ureter was 32.1 mm (7.5-46.1). Neither major vessels nor the ureter was injured. Dissection of the hypogastric plexus was undertaken in 14 cadavers, and in each cadaver, tacks affected the hypogastric nerve plexus. LIMITATIONS: This study was limited by the moderate number of cadavers. CONCLUSIONS: Tack placement showed significant variation in our specimen, emphasising the need for reliable anatomic landmarks and sufficient exposure during ventral mesh rectopexy. Hypogastric nerve plexus involvement is common, thus detailed functional assessment after surgery is required. It also points out the importance of cadaver studies before implementing new surgical techniques into clinical practice. See Video Abstract at http://links.lww.com/DCR/B827. FIJACIN LAPAROSCPICA DE MALLA SACRA PARA RECTOPEXIA VENTRAL IMPLICACIONES CLNICAS DE UN ESTUDIO SOBRE CADAVERS: ANTECEDENTES:La colocación correcta de la tachuela en el promontorio sacro para la fijación de la malla en la rectopexia con malla ventral es crucial para evitar hemorragias, disfunción nerviosa y espondilodiscitis.OBJETIVO:El presente estudio en cadáveres fue diseñado para evaluar la verdadera ubicación de las tachuelas después de la fijación de la malla durante la rectopexia laparoscópica con malla ventral en relación con las estructuras vasculares y nerviosas y los puntos de referencia óseos.DISEÑO:Estudio intervencionista de cadáveres.AJUSTE:Después de la fijación laparoscópica de la malla, se realizó una disección pélvica detallada siguiendo un protocolo estandarizado. Además, se realizó una tomografía computarizada multidetector de 64 cortes para definir mejor la anatomía lumbosacra y la posición de la tachuela.PACIENTES:Se incluyeron en este estudio dieciocho cadáveres frescos (10 mujeres, 8 hombres).PRINCIPALES MEDIDAS DE RESULTADO:Posición real de tachuela y compromiso vascular y neuronal.RESULTADOS:Se utilizaron un total de 52 tachuelas (mediana 3, 2-3 tachuelas). La distancia media de tachuela al promontorio sacro medio fue de 16,1 mm (0,0-54,2). Solo se encontraron un total de 22 tachuelas (42,3%) en la superficie derecha de la vértebra S1, correlacionándose con el área planificada. En siete cadáveres (38,8%) todas las tachuelas se utilizaron en el área de planificada. La distancia media a los vasos principales fue de 10,5 mm (0,0-35,0), que era la arteria ilíaca interna en la mitad de los casos. La distancia media de las tachuelas al uréter derecho fue de 32,1 mm (7,5-46,1). No se lesionó ni los grandes vasos ni el uréter. La disección del plexo hipogástrico se realizó en 14 cadáveres y en cada cadáver, las tachuelas afectaron el plexo nervioso hipogástrico.LIMITACIONES:Número moderado de cadáveres incluidos en el estudio.CONCLUSIONES:La colocación de tachuelas mostró una variación significativa en nuestra muestra, enfatizando la necesidad de puntos de referencia anatómicos confiables y una exposición suficiente durante la rectopexia con malla ventral. La afectación del plexo nervioso hipogástrico es común, por lo que se requiere una evaluación funcional detallada después de la cirugía. También destaca la importancia de los estudios sobre cadáveres antes de implementar nuevas técnicas quirúrgicas en la práctica clínica. Consulte Video Resumen en http://links.lww.com/DCR/B827. (Traducción-Dr Yolanda Colorado).

Anatomic basics and technical approaches: sacral preauricular extensions, preauricular sulci and dorsal pubic pits in modern anatomical specimens.

The aim of this study is the evaluation of three selected osseous pelvic features in modern anatomical specimen - the sacral preauricular extension, the preauricular sulcus and pits on the dorsal side of the pubic bone laterally to the symphysis. The specificity and significance of these features are under debate and their genesis is largely unclear. Descriptive data of specific soft tissue structures surrounding the anterior sacroiliac joint gap and the pubic symphysis were generated by assessing 20 fresh pelves and 12 embalmed hemipelves from human body donors. Computed tomography (CT) was performed on all specimens and three-dimensional (3D) surface models were generated and analysed. Afterwards, all the specimens underwent anatomical dissection and finally maceration. During dissection, it became apparent that the anterior sacroiliac ligament, due to its position, shape and potential impact on the sacroiliac joint and adjacent osseous structures, requires a detailed analysis of its dimension. The most promising result, in terms of the sacral preauricular extension, was that the measurements of the triangular part of the anterior sacroiliac ligament were significantly longer in females than in males. Pelvic floor muscle fibres and fascial parts were directly connected to this ligament in some specimens, which is an important starting point for a larger in-depth study. The evaluation of the anatomic structures in connection to dorsal pitting refutes the assumption that the pelvic floor muscles or fasciae could exert influence on its formation. A histological evaluation of the ligaments of the pubic symphysis, especially the dorsal pubic ligament, would be expedient to clarify the etiology of this feature.

The Col4a2em1(IMPC)Wtsi mouse line: lessons from the Deciphering the Mechanisms of Developmental Disorders program.

The Deciphering the Mechanisms of Developmental Disorders (DMDD) program uses a systematic and standardised approach to characterise the phenotype of embryos stemming from mouse lines, which produce embryonically lethal offspring. Our study aims to provide detailed phenotype descriptions of homozygous Col4a2em1(IMPC)Wtsi mutants produced in DMDD and harvested at embryonic day 14.5. This shall provide new information on the role Col4a2 plays in organogenesis and demonstrate the capacity of the DMDD database for identifying models for researching inherited disorders. The DMDD Col4a2em1(IMPC)Wtsi mutants survived organogenesis and thus revealed the full spectrum of organs and tissues, the development of which depends on Col4a2 encoded proteins. They showed defects in the brain, cranial nerves, visual system, lungs, endocrine glands, skeleton, subepithelial tissues and mild to severe cardiovascular malformations. Together, this makes the DMDD Col4a2em1(IMPC)Wtsi line a useful model for identifying the spectrum of defects and for researching the mechanisms underlying autosomal dominant porencephaly 2 (OMIM # 614483), a rare human disease. Thus we demonstrate the general capacity of the DMDD approach and webpage as a valuable source for identifying mouse models for rare diseases.

A Specific CNOT1 Mutation Results in a Novel Syndrome of Pancreatic Agenesis and Holoprosencephaly through Impaired Pancreatic and Neurological Development.

We report a recurrent CNOT1 de novo missense mutation, GenBank: NM_016284.4; c.1603C>T (p.Arg535Cys), resulting in a syndrome of pancreatic agenesis and abnormal forebrain development in three individuals and a similar phenotype in mice. CNOT1 is a transcriptional repressor that has been suggested as being critical for maintaining embryonic stem cells in a pluripotent state. These findings suggest that CNOT1 plays a critical role in pancreatic and neurological development and describe a novel genetic syndrome of pancreatic agenesis and holoprosencephaly.

Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM).

The article will briefly introduce the high-resolution episcopic microscopy (HREM) technique and will focus on its potential for researching cardiovascular development and remodelling in embryos of biomedical model organisms. It will demonstrate the capacity of HREM for analysing the cardiovascular system of normally developed and genetically or experimentally malformed zebrafish, frog, chick and mouse embryos in the context of the whole specimen and will exemplarily show the possibilities HREM offers for comprehensive visualisation of the vasculature of adult human skin. Finally, it will provide examples of the successful application of HREM for identifying cardiovascular malformations in genetically altered mouse embryos produced in the deciphering the mechanisms of developmental disorders (DMDD) program.

Comparative study of regenerative effects of mesenchymal stem cells derived from placental amnion, chorion and umbilical cord on dermal wounds.

OBJECTIVE: Mesenchymal stem/stromal cells derived from human term placentas (PMSCs) are novel therapeutic agents and more topical than ever. Here we evaluated the effects of three types of PMSCs on wound healing in an in vivo mouse model: Amnion-derived MSCs (AMSCs), blood vessel-derived MSCs (BV-MSCs) from the chorionic plate and Wharton's jelly-derived MSCs (WJ-MSCs) from the umbilical cord. METHODS: We topically applied PMSCs onto skin wounds in mice using the dermal substitute Matriderm® as carrier and evaluated wound healing parameters. In addition, we investigated the effects of all PMSC types under co-application with placental endothelial cells (PLECs). After 8 days, we compared the percent of wound closure and the angiogenic potential between all groups. RESULTS: AMSCs, BV-MSCs and WJ-MSCs significantly induced a faster healing and a higher number of blood vessels in the wound when compared to controls (Matriderm®-alone). PLECs did not further improve the advantageous effects of PMSC-treatment. Quantitative data and 3D analysis by high resolution episcopic microscopy confirmed a lower density of vessels in Matriderm®/PMSCs/PLECs co-application compared to Matriderm®/PMSCs treatment. CONCLUSION: Results indicate that all three PMSC types exert similar beneficial effects on wound closure and neovascularization in our mouse model. PRACTICE: Using Matriderm® as carrier for PMSCs propagates rapid cell migration towards the wound area that allows a fast and clinically practicable method for stem cell application. IMPLICATIONS: These promising effects warrant further investigation in clinical trials.

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials.

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging organic materials with volumes up to 5 x 5 x 7 mm3 in typical numeric resolutions between 1 x 1 x 1 and 5 x 5 x 5 µm3. Specimens are embedded in methacrylate resin and sectioned on a microtome. After each section an image of the block surface is captured with a digital video camera that sits on the phototube connected to the compound microscope head. The optical axis passes through a green fluorescent protein (GFP) filter cube and is aligned with a position, at which the bock holder arm comes to rest after each section. In this way, a series of inherently aligned digital images, displaying subsequent block surfaces are produced. Loading such an image series in three-dimensional (3D) visualization software facilitates the immediate conversion to digital volume data, which permit virtual sectioning in various orthogonal and oblique planes and the creation of volume and surface rendered computer models. We present three simple, tissue specific protocols for processing various groups of organic specimens, including mouse, chick, quail, frog and zebra fish embryos, human biopsy material, uncoated paper and skin replacement material.

Palisade Endings Are a Constant Feature in the Extraocular Muscles of Frontal-Eyed, But Not Lateral-Eyed, Animals.

PURPOSE: To test whether palisade endings are a general feature of mammalian extraocular muscles (EOMs). METHODS: Thirteen species, some frontal-eyed (human, monkey, cat, and ferret), and others lateral-eyed (pig, sheep, calf, horse, rabbit, rat, mouse, gerbil, and guinea pig) were analyzed. Palisade endings were labeled by using different combinations of immunofluorescence techniques. Three-dimensional reconstructions of immunolabeled palisade endings were done. RESULTS: In all frontal-eyed species, palisade endings were a consistent feature in the rectus EOMs. Their total number was high and they exhibited an EOM-specific distribution. In particular, the number of palisade endings in the medial recti was significantly higher than in the other rectus muscles. In the lateral-eyed animals, palisade endings were infrequent and, when present, their total number was rather low. They were only found in ungulates (sheep, calf, pig, and horse) and in rabbit. In rodents (rat, guinea pig, mouse, and gerbil) palisade endings were found infrequently (e.g., rat) or were completely absent. Palisade endings in frontal-eyed species and in some lateral-eyed species (pig, sheep, calf, and horse) had a uniform morphology. They generally lacked α-bungarotoxin staining, with a few exceptions in primates. Palisade endings in other lateral-eyed species (rabbit and rat) exhibited a simplified morphology and bound α-bungarotoxin. CONCLUSIONS: Palisade endings are not a universal feature of mammalian EOMs. So, if they are proprioceptors, not all species require them. Because in frontal-eyed species, the medial rectus muscle has the highest number of palisade endings, they likely play a special role in convergence.