Spatial transcriptomics in embryonic mouse diaphragm muscle reveals regional gradients and subdomains of developmental gene expression.

The murine embryonic diaphragm is a primary model for studying myogenesis and neuro-muscular synaptogenesis, both representing processes regulated by spatially organized genetic programs of myonuclei located in distinct myodomains. However, a spatial gene expression pattern of embryonic mouse diaphragm has not been reported. Here, we provide spatially resolved gene expression data for horizontally sectioned embryonic mouse diaphragms at embryonic days E14.5 and E18.5. These data reveal gene signatures for specific muscle regions with distinct maturity and fiber type composition, as well as for a central neuromuscular junction (NMJ) and a peripheral myotendinous junction (MTJ) compartment. Comparing spatial expression patterns of wild-type mice with those of transgenic mice lacking either the skeletal muscle calcium channel CaV1.1 or ß-catenin, reveals curtailed muscle development and dysregulated expression of genes potentially involved in NMJ formation. Altogether, these datasets provide a powerful resource for further studies of muscle development and NMJ formation in the mouse.

Cadaveric study of ergonomics and performance using a robotic exoscope with a head-mounted display in spine surgery.

The conventional microscope has the disadvantage of a potentially unergonomic posture for the surgeon, which can affect performance. Monitor-based exoscopes could provide a more ergonomic posture, as already shown in pre-clinical studies. The aim of this study was to test the usability and comfort of a novel head-mounted display (HMD)-based exoscope on spinal surgical approaches in a simulated OR setting. A total of 21 neurosurgeons naïve to the device were participated in this prospective trial. After a standardized training session with the device, participants were asked to perform a single-level thoracolumbar decompression surgery on human cadavers using the exoscope. Subsequently, all participants completed a comfort and safety questionnaire. For the objective evaluation of the performance, all interventions were videotaped and analyzed. Twelve men and nine women with a mean age of 34 (range: 24-57) were participating in the study. Average time for decompression was 15 min (IqR 9.6; 24.2); three participants (14%) terminated the procedure prematurely. In these dropouts, a significantly higher incidence of back/neck pain (p = 0.002 for back, p = 0.046 for neck pain) as well as an increased frequency of HMD readjustments (p = 0.045) and decreased depth perception (p = 0.03) were documented. Overall, the surgeons' satisfaction with the exoscope was 84% (IqR 75; 100). Using a standardized, pre-interventional training, it is possible for exoscope-naïve surgeons to perform sufficient spinal decompression using the HMD-based exoscope with a high satisfaction. However, inaccurate HMD setup prior to the start of the procedure may lead to discomfort and unsatisfactory results.

The relationship between connexin-43 expression and Ki67 in non-glial central nervous system tumors.

BACKGROUND: Advanced intercellular communication is a known oncogenic factor. In the central nervous system, Connexin-43 (Cx43) forms this junctional networking. Moreover, it correlates with the proliferation rate, and thus behavior, of gliomas. We assessed the expression of Cx43 and its relationship to Ki67 in other common central nervous system tumors. METHODS: The expression of Cx43 and Ki67 were assessed in formalin-fixed paraffin embedded samples of human brain metastases, meningiomas, and neurinomas using immunohistochemistry. Neurinomas and meningiomas were jointly evaluated due to similar non-malignant behavior. RESULTS: A total of 14 metastases of different extracerebral carcinomas, 6 meningiomas, and 10 neurinomas were evaluated. Five (36%) metastases and 5 (31%) meningiomas/neurinomas showed minor expression, whereas 6 (43%) metastases and 2 (13%) meningiomas/neurinomas showed no Cx43 expression at all. In 3 (21%) metastases and 9 (56%) meningiomas/neurinomas, moderate or strong expression of Cx43 was identified. The higher expression of Cx43 in meningiomas and neurinomas directly correlated with Ki67, r = 0.53 (P = 0.034). For metastases no significant correlation was found. Mitotic index in meningiomas/neurinomas correlated with Ki67 expression, r = 0.74 (P < 0.001), but did not show statistically significant correlation with Cx43 expression in these tumors. CONCLUSIONS: The expression of Cx43 as a marker of cell-to-cell networking exposed a significant correlation with the Ki67-defined proliferation index in case of primary central nervous system neuroectodermal neoplasms. However, it does not seem to play a comparable role in metastases with extracerebral origin.

Impact of GAP-43, Cx43 and actin expression on the outcome and overall survival in diffuse and anaplastic gliomas.

Distant intercellular communication in gliomas is based on the expansion of tumor microtubuli, where actin forms cytoskeleton and GAP-43 mediates the axonal conus growth. We aimed to investigate the impact of GAP-43 and actin expression on overall survival (OS) as well as crucial prognostic factors. FFPE tissue of adult patients with diffuse and anaplastic gliomas, who underwent first surgery in our center between 2010 and 2019, were selected. GAP-43, Cx43 and actin expression was analyzed using immunohistochemistry and semi-quantitatively ranked. 118 patients with a median age of 46 years (IqR: 35-57) were evaluated. 48 (41%) presented with a diffuse glioma and 70 (59%) revealed anaplasia. Tumors with higher expression of GAP-43 (p = 0.024, HR = 1.71/rank) and actin (p < 0.001, HR = 2.28/rank) showed significantly reduced OS. IDH1 wildtype glioma demonstrated significantly more expression of all proteins: GAP-43 (p = 0.009), Cx43 (p = 0.003) and actin (p < 0.001). The same was confirmed for anaplasia (GAP-43 p = 0.028, actin p = 0.029), higher proliferation rate (GAP-43 p = 0.016, actin p = 0.038), contrast-enhancement in MRI (GAP-43 p = 0.023, actin p = 0.037) and age (GAP-43 p = 0.004, actin p < 0.001; Cx43 n.s. in all groups). The intercellular distant communication network in diffuse and anaplastic gliomas formed by actin and GAP-43 is associated with a negative impact on overall survival and with unfavorable prognostic features. Cx43 did not show relevant impact on OS.

The distance between the distal margin of the flexor retinaculum and the radiocarpal joint as a landmark in CTS surgery: A cadaver study.

BACKGROUND: The most frequent peripheral entrapment neuropathy is compression of the median nerve in the carpal tunnel, known as carpal tunnel syndrome. The most effective treatment is surgery, where the flexor retinaculum (FR) is divided. Nevertheless, after this operation, a significant number of patients suffer from persistent symptoms due to incomplete FR distal release. It may be difficult to identify the distal boundary of the FR due to the minimal skin incision. We aimed to identify an anatomical landmark to avoid incomplete distal FR release. The radiocarpal (RC) joint can be palpated, and lies in close proximity to the boundaries of the FR. Thus, the distance between the RC joint space and the distal FR margin - the RC-FR distance - could be a reliable and individual morphologic measurement from easily acquired regional anthropological measurements. METHODS: During this study, 39 radiocarpal regions of 23 embalmed cadavers were dissected, and measurements were taken. Linear regression corresponding to the ulnar length and the RC-FR distance was established. RESULTS: The mean RC-FR distance from the RC joint space to the distal FR margin was 3.8 cm (95 % CI 3.5-4.0), and the range was 2.3-5.1 cm. This distance was 1.1 cm (95 % CI 0.8-1.4) longer in males than in females (p < 0.00001), and there were no side-specific differences. The individual projection of the distal FR margin in centimeters can be calculated by measurement of the ipsilateral ulnar length divided by 4 and reduced by 2.9, p < 0.005. CONCLUSIONS: The side-equal and sex-specific position of the distal flexor retinaculum margin could be calculated from the palpable radiocarpal joint space based on the ipsilateral ulnar length.

Surgeon's comfort: The ergonomics of a robotic exoscope using a head-mounted display.

Introduction: Conventional microscopes have certain limitations in terms of posture and ergonomics. Monitor-based exoscopes could solve this problem and thereby lead to less work-related sick leave for surgeons. Research question: The aim of this study was to assess the ergonomics, usability, and neurosurgeon's comfort of a novel three-dimensional head-mounted display-based exoscope in a standardized setting. Material & Methods: 34 neurosurgeons participated in a workshop on the exoscope, which features a head-mounted display and a head gesture-triggered control panel. After completion of a custom-made 10-step microsurgical exercise, image quality and comfort were assessed using a questionnaire. The participants' posture during the exercise was analyzed using a video motion analysis software. Results: 34 participants (median neurosurgical experience: 6 years) were included. The median time to complete the exercise was 12 â€‹min [IqR 9.4, 15.0]. Younger participants (p â€‹= â€‹0.005) and those with video game experience (p â€‹= â€‹0.03) had a significantly steeper learning curve. The median overall satisfaction was at 80% in general and 82% for image quality. The median upper body as well as the median head coronal displacement from the neutral axis were 0°. Participants with less microsurgical experience showed less head/body displacement during the exercise (p â€‹= â€‹0.01). Discussion and conclusion: Using the microsurgical training tool, we were able to depict a steep learning curve with a sufficient learnability of the most relevant commands. The exoscope excelled in usability, image quality as well as in ergonomic and favorable posture and could thus become an alternative to conventional microscopes due to the potentially elevated surgeons' comfort.

A new method of intraoperative pelvic neuromonitoring: a preclinical feasibility study in a porcine model.

Low anterior resections (LAR) are frequently associated with complications such as urinary and fecal incontinence as well as sexual disorders. Typical risk factors are rectal cancer with low tumor location, preoperative radiotherapy, and surgery-related damage of pelvic autonomic nerves. As preserving the pelvic autonomic nerves without any technical assistance is challenging, the objective of this preclinical study was to investigate the technical feasibility of a new method for intraoperative pelvic neuromonitoring. Twelve female pigs undergoing low anterior resections were involved in a prospective preclinical study. Intraoperative pelvic neuromonitoring included direct pelvic nerve stimulation and tissue impedance measurement on the urinary bladder and the rectum for the identification of efferent pelvic nerves in the surgical area. Immunohistochemistry was used to verify the results. Smooth muscle contraction of the urinary bladder and/or the rectum in response to direct stimulation of the innervating nerves was detectable with impedance measurement. The macroscopic contraction of both the urinary bladder and the rectum correlated with a change in tissue impedance compared to the status before contraction. Thus, it was possible to identify pelvic nerves in the surgical area, which allows the nerves to be preserved. The results indicate a reliable identification of pelvic autonomic nerves, which allows nerve damage to be prevented in the future.

The advanced development of Cx43 and GAP-43 mediated intercellular networking in IDH1 wildtype diffuse and anaplastic gliomas with lower mitotic rate.

PURPOSE: The biologic behavior and the therapeutic resistance of diffuse and anaplastic gliomas varies greatly. This may be explained by differences in cell-to-cell communication, determined by the Cx43-associated junctional activity and the microtubules-defined network, in which GAP-43 is the dominant structural component. We assessed the expression of these crucial communication proteins in samples of patients harboring WHO°II and III gliomas, graded according to the current 4th revised WHO classification. METHODS: Tissue of adult patients with WHO°II and III gliomas, who underwent surgery between 2014 and 2018, were selected from our institutional biobank. GAP-43 and Cx43 expression was analyzed using IHC. Routine clinical and neuropathological findings were additionally retrieved from our institutional prospective database. RESULTS: 43 (57%) males and 33 (43%) females with a median age of 47 (IqR: 35-61) years were selected. IDH1 wildtype tumors showed a significantly higher expression of Cx43 (p = 0.014) and a tendency for increased GAP-43 production. Advanced Cx43 expression significantly correlated with lower mitosis rate (p = 0.014): more in IDH1 wildtype (r = - 0.57, p = 0.003) than in mutated gliomas (r = - 0.37, p = 0.019). There was no difference in Cx43 or GAP-43 expression in relation to anaplastic phenotype, Gadolinum-contrasted enhancement (CE) on MRI and advanced EGFR or p53 expression. CONCLUSIONS: Intercellular communication tends to be more relevant in slower proliferating, e.g. lower malignant tumors. They could have more time to establish this network, providing longitudinally acquired resistance against specific oncological therapy. This feature matches the unfavorable IDH1 wildtype status of glioma and supports the noted malignant behavior of these tumors in the upcoming 5th WHO classification of gliomas.

The development of the human vaginal fornix and the portio cervicis.

INTRODUCTION: One of the transitional zones of the human body is situated in the cervix uteri. The developmental differentiation of epithelial and stromal characteristics in such a region is of high clinical interest. However, few studies have focused on the development of this region, and information in anatomical and clinical textbooks is limited. We therefore examined the development of the human vaginal fornix and the cervix uteri during prenatal development. MATERIALS AND METHODS: We examined 29 female embryos and fetuses between 20 and 34 weeks and two newborns using histology and immunohistochemistry. RESULTS: The characteristic shape of the portiocervicis and the vaginal fornix first became visible in mid-term fetuses because of the different muscular coats and of an uncategorized Müllerian-derived epithelium, which was rapidly replaced by a multilayered squamous epithelium. Only thereafter, in older fetuses, were there organogenetic differentiation of the epithelia and the underlying stroma of the cervical canal. UGS-derived p63/CK17-positive cells could be identified as precursor cells for the squamous epithelium, and Müllerian-derived CK7-positive cells for the columnar-type epithelium. Both cell types and different stromal zones were already present in a prenatal transformation zone. Initial functional differentiation could be observed in perinatal stages. CONCLUSIONS: Our results on prenatal human development strongly support the view that two different cell lineages meet at the transitional zone of the cervix uteri and that these lineages depend on alternative signals from the underlying stromal compartment.

Defining a therapeutic range for regeneration of ischemic myocardium via shock waves.

Shockwave therapy (SWT) represents a promising regenerative treatment option for patients with ischemic cardiomyopathy. Although no side-effects have been described upon SWT, potential cellular damage at therapeutic energies has not been addressed so far. In this work, we aimed to define a therapeutic range for shock wave application for myocardial regeneration. We could demonstrate that SWT does not induce cellular damage beneath energy levels of 0.27 mJ/mm2 total flux density. Endothelial cell proliferation, angiogenic gene expression and phosphorylation of AKT and ERK are enhanced in a dose dependent manner until 0.15 mJ/mm2 energy flux density. SWT induces regeneration of ischemic muscle in vivo via expression of angiogenic gene expression, enhanced neovascularization and improved limb perfusion in a dose-dependent manner. Therefore, we provide evidence for a dose-dependent induction of angiogenesis after SWT, as well as the absence of cellular damage upon SWT within the therapeutic range. These data define for the first time a therapeutic range of SWT, a promising regenerative treatment option for ischemic cardiomyopathy.

Shock waves promote spinal cord repair via TLR3.

Spinal cord injury (SCI) remains a devastating condition with poor prognosis and very limited treatment options. Affected patients are severely restricted in their daily activities. Shock wave therapy (SWT) has shown potent regenerative properties in bone fractures, wounds, and ischemic myocardium via activation of the innate immune receptor TLR3. Here, we report on the efficacy of SWT for regeneration of SCI. SWT improved motor function and decreased lesion size in WT but not Tlr3-/- mice via inhibition of neuronal degeneration and IL6-dependent recruitment and differentiation of neuronal progenitor cells. Both SWT and TLR3 stimulation enhanced neuronal sprouting and improved neuronal survival, even in human spinal cord cultures. We identified tlr3 as crucial enhancer of spinal cord regeneration in zebrafish. Our findings indicate that TLR3 signaling is involved in neuroprotection and spinal cord repair and suggest that TLR3 stimulation via SWT could become a potent regenerative treatment option.

miR-19a-3p containing exosomes improve function of ischaemic myocardium upon shock wave therapy.

AIMS: As many current approaches for heart regeneration exert unfavourable side effects, the induction of endogenous repair mechanisms in ischaemic heart disease is of particular interest. Recently, exosomes carrying angiogenic miRNAs have been described to improve heart function. However, it remains challenging to stimulate specific release of reparative exosomes in ischaemic myocardium. In the present study, we sought to test the hypothesis that the physical stimulus of shock wave therapy (SWT) causes the release of exosomes. We aimed to substantiate the pro-angiogenic impact of the released factors, to identify the nature of their cargo, and to test their efficacy in vivo supporting regeneration and recovery after myocardial ischaemia. METHODS AND RESULTS: Mechanical stimulation of ischaemic muscle via SWT caused extracellular vesicle (EV) release from endothelial cells both in vitro and in vivo. Characterization of EVs via electron microscopy, nanoparticle tracking analysis and flow cytometry revealed specific exosome morphology and size with the presence of exosome markers CD9, CD81, and CD63. Exosomes exhibited angiogenic properties activating protein kinase b (Akt) and extracellular-signal regulated kinase (ERK) resulting in enhanced endothelial tube formation and proliferation. A miRNA array and transcriptome analysis via next-generation sequencing were performed to specify exosome content. miR-19a-3p was identified as responsible cargo, antimir-19a-3p antagonized angiogenic exosome effects. Exosomes and target miRNA were injected intramyocardially in mice after left anterior descending artery ligation. Exosomes resulted in improved vascularization, decreased myocardial fibrosis, and increased left ventricular ejection fraction as shown by transthoracic echocardiography. CONCLUSION: The mechanical stimulus of SWT causes release of angiogenic exosomes. miR-19a-3p is the vesicular cargo responsible for the observed effects. Released exosomes induce angiogenesis, decrease myocardial fibrosis, and improve left ventricular function after myocardial ischaemia. Exosome release via SWT could develop an innovative approach for the regeneration of ischaemic myocardium.

Early appearance of key transcription factors influence the spatiotemporal development of the human inner ear.

Expression patterns of transcription factors leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), transforming growth factor-ß-activated kinase-1 (TAK1), SRY (sex-determining region Y)-box 2 (SOX2), and GATA binding protein 3 (GATA3) in the developing human fetal inner ear were studied between the gestation weeks 9 and 12. Further development of cochlear apex between gestational weeks 11 and 16 (GW11 and GW16) was examined using transmission electron microscopy. LGR5 was evident in the apical poles of the sensory epithelium of the cochlear duct and the vestibular end organs at GW11. Immunostaining was limited to hair cells of the organ of Corti by GW12. TAK1 was immune positive in inner hair cells of the organ of Corti by GW12 and colocalized with p75 neurotrophic receptor expression. Expression for SOX2 was confined primarily to the supporting cells of utricle at the earliest stage examined at GW9. Intense expression for GATA3 was presented in the cochlear sensory epithelium and spiral ganglia at GW9. Expression of GATA3 was present along the midline of both the utricle and saccule in the zone corresponding to the striolar reversal zone where the hair cell phenotype switches from type I to type II. The spatiotemporal gradient of the development of the organ of Corti was also evident with the apex of the cochlea forming by GW16. It seems that highly specific staining patterns of several transcriptions factors are critical in guiding the genesis of the inner ear over development. Our findings suggest that the spatiotemporal gradient in cochlear development extends at least until gestational week 16.

FGF8, FGF10 and FGF receptor 2 in foreskin of children with hypospadias: an analysis of immunohistochemical expression patterns and gene transcription.

INTRODUCTION: Fibroblast growth factors (FGFs) play a crucial role in early embryogenesis of the genital tubercle and are involved in the development of hypospadias, affecting both endo- and ectodermally derived tissues. It was hypothesized that expression of FGFs could be qualitatively or quantitatively altered in skin of children with hypospadias. OBJECTIVE: The objective of the study was to investigate expression patterns and transcription levels of FGF8, FGF10, and FGF Receptor 2 (FGFR2) in patients with hypospadias compared to normal controls. PATIENTS AND METHODS: Skin samples from the ventro-lateral aspect of the foreskin of 32 patients with hypospadias (17 distal and 15 proximal, mean age 25 months) and 10 normal foreskin samples (mean age 77 months) were analyzed by immunohistochemistry. Staining, localization, and distribution of positive cells in epidermis and dermis were categorized independently by two researchers. Complementary DNA (cDNA) samples prepared from messenger RNA (mRNA) isolates of the same samples were analyzed by quantitative polymerase chain reaction (qPCR), comparing expressions of FGF8, FGF10, and FGFR2 with loading controls. RESULTS: Patients with hypospadias consistently showed aberrant immunohistochemical staining patterns for FGF8/FGF10/FGFR2 in epidermis and dermis compared to patients without penile malformation (p < 0.01 for all markers). qPCR displayed no difference in expression levels on mRNA level (FGFR2 p = 0.44, FGF8 p = 0.77, and FGF10 p = 0.17) comparing normal foreskin with foreskin from patients with hypospadias. Figure. DISCUSSION: The results point at an impact of FGF signaling during embryological development of hypospadias on skin, as an ectodermally derived tissue. Similar to the urethral development, this might be a result of mesothelial-epithelial interactions. The differing expression patterns in immunohistochemistry are not matched by a quantitative difference in marker expression on the mRNA level, putatively caused by post-translational modifications or alterations of the downstream pathway. FGFs, particularly FGF10 and FGFR2, are critically involved in wound healing. CONCLUSIONS: There are significant differences in localization and distribution of FGF8, FGF10, and FGFR2 in comparisons of normal foreskin to foreskin of patients with hypospadias, whereas there is no difference in the quantitative expression of these markers on the mRNA level. This confirms the notion that penile skin is affected as well by the embryological aberrations during the embryogenesis of hypospadias.

Postoperative Hypoparathyroidism in Thyroid Surgery: Anatomic-Surgical Mapping of the Parathyroids and Implications for Thyroid Surgery.

Hypoparathyroidism remains one of the most common complications in thyroid surgery. This study aims for an improved understanding of the complexity of the blood supply and the localisation of the parathyroids compared to the two most important intraoperative landmarks: the inferior laryngeal nerve (ILN) and Zuckerkandl's tubercle (ZT). We examined 103 laryngeal compounds to classify the blood supply and the localisation of the parathyroids. For intraoperative localisation we defined a Cartesian coordinate system with the ZT plane as x-axis and the course of the inferior laryngeal nerve as y-axis. The inferior thyroid artery (ITA) mainly supplies the parathyroids, whereas the superior thyroid artery provides a backup supply. It must be pointed out that 8.2% of parathyroids receive their blood directly from the thyroid gland. 73.5% of all parathyroids lie within 1 cm of the ILN and 1 cm cranial and 2.5 cm caudal to the ZT plane. Our described perimeters mark the most crucial areas during surgery and provide the surgeon with an anatomic mapping showing areas of special carefulness needed. One should keep bearing in mind all possible blood supply types of the parathyroids and therefore all branches should be handled with care.

Langerhans cells in hypospadias: an analysis of Langerin (CD207) and HLA-DR on epidermal sheets and full thickness skin sections.

BACKGROUND: Hypospadias are among the most common genital malformations. Langerhans Cells (LCs) play a pivotal role in HIV and HPV infection. The migration of LC precursors to skin coincides with the embryonic period of hypospadias development and genetic alterations leading to the formation of hypospadias impact the development of ectodermally derived tissues. We hypothesized that this might be associated with a difference in frequency or morphology of epidermal and dermal LCs in hypospadias patients. METHODS: A total of 43 patients from two centers were prospectively included into this study after parental consent and ethics approval. Epidermal and dermal sheets were prepared from skin samples of 26 patients with hypospadias, 13 patients without penile malformations and 4 patients with penile malformations other than hypospadias. Immunofluorescence staining of sheets was performed with anti-HLA-DR-FITC and anti-CD207/Langerin-A594 antibodies. Skin sections from 11 patients without penile malformation and 11 patients with hypospadias were stained for Langerin. Frequencies as well as morphology and distribution of epidermal and dermal LCs on sheets and sections were microscopically evaluated. Cell counts were compared by unpaired t-tests. RESULTS: There was no difference in frequency of epidermal LCs, Neither on sheets (873 ± 61 vs. 940 ± 84LCs/mm2, p = 0.522) nor on sections (32 ± 3 vs. 30 ± 2LCs/mm2, p = 0.697). Likewise, the frequency of dermal LCs (5,9 ± 0,9 vs. 7.5 ± 1.3LCs/mm2, p = 0.329) was comparable between patients with hypospadias and without penile malformation. No differences became apparent in subgroup analyses, comparing distal to proximal hypospadias (p = 0.949), younger and older boys (p = 0.818) or considering topical dihydrotestosterone treatment prior to surgery (p = 0.08). The morphology of the LCs was not different comparing hypospadias patients with boys without penile malformations. CONCLUSIONS: LCs are present in similar frequencies and with a comparable morphology and distribution in patients with hypospadias as compared to children without penile malformations. This suggests that patients with hypospadias are not different from patients with normal penile development considering this particular compartment of their skin immunity.

Chemical imaging and assessment of cadmium distribution in the human body.

The scientific interest in cadmium (Cd) as a human health damaging agent has significantly increased over the past decades. However, particularly the histological distribution of Cd in human tissues is still scarcely defined. Using inductively coupled plasma-mass spectrometry (ICP-MS), we determined the concentration of Cd in 40 different human tissues of four body donors and provided spatial information by elemental imaging on the microscopic distribution of Cd in 8 selected tissues by laser ablation (LA)-ICP-MS. ICP-MS results show that Cd concentrations differ by a factor of 20 000 between different tissues. Apart from the well know deposits in kidney, bone, and liver, our study provides evidence that muscle and adipose tissue are underestimated Cd pools. For the first time, we present spatially resolved Cd distributions in a broad panel of human soft tissues. The defined histological structures are mirrored by sharp cut differences in Cd concentrations between neighboring tissue types, particularly in the rectum, testis, and kidneys. The spatial resolution of the Cd distribution at microscopic level visualized intratissue hot spots of Cd accumulation and is suggested as a powerful tool to elucidate metal based toxicity at histological level.

Growth and cellular patterning during fetal human inner ear development studied by a correlative imaging approach.

BACKGROUND: Progressive transformation of the otic placode into the functional inner ear during gestational development in humans leads to the acquisition of hearing perception via the cochlea and balance and spatial orientation via the vestibular organ. RESULTS: Using a correlative approach involving micro-computerized tomography (micro-CT), transmission electron microscopy and histological techniques we were able to examine both the morphological and cellular changes associated with human inner ear development. Such an evaluation allowed for the examination of 3D geometry with high spatial and temporal resolution. In concert with gestational progression and growth of the cochlear duct, an increase in the distance between some of the Crista ampullaris is evident in all the specimens examined from GW12 to GW36. A parallel increase in the distances between the macular organs - fetal utricle and saccule - is also evident across the gestational stages examined. The distances between both the utricle and saccule to the three cristae ampullares also increased across the stages examined. A gradient in hair cell differentiation is apparent from apex to base of the fetal cochlea even at GW14. CONCLUSION: We present structural information on human inner ear development across multiple levels of biological organization, including gross-morphology of the inner ear, cellular and subcellular details of hearing and vestibular organs, as well as ultrastructural details in the developing sensory epithelia. This enabled the gathering of detailed information regarding morphometric changes as well in realizing the complex developmental patterns of the human inner ear. We were able to quantify the volumetric and linear aspects of selected gestational inner ear specimens enabling a better understanding of the cellular changes across the fetal gestational timeline. Moreover, these data could serve as a reference for better understanding disorders that arise during inner ear development.

Shock wave treatment after hindlimb ischaemia results in increased perfusion and M2 macrophage presence.

Shock wave therapy (SWT) has been shown to induce angiogenesis in ischaemic muscle. However, the mechanism of action remains unknown. Macrophages are crucial for angiogenic responses after ischaemic injury. The M2 macrophage subset enables tissue repair and induces angiogenesis. It was hypothesized that the angiogenic effects of SWT are at least partly caused by enhanced macrophage recruitment. C57BL/6 mice were subjected to hind limb ischaemia with subsequent SWT or sham treatment. Muscles were analysed via immunofluorescence staining, reverse-transcription polymerase chain reaction and western blot. Gene expression and proteins involved in macrophage recruitment were analysed and tissue sections were stained for macrophages, including subsets, capillaries and arterioles. Laser Doppler perfusion imaging was performed to assess functional outcome. Treated muscles showed increased expression of the pivotal macrophage recruiting factor monocyte chemotactic protein 1 (MCP-1). Higher levels of macrophage marker CD14 were found. Increased numbers of macrophages after SWT could be confirmed by immunofluorescence staining. The expression of the M2 polarization promoting chemokine interleukin 13 was significantly elevated in the treatment group. Elevated mRNA expression of the M2 scavenger receptor CD163 was found after SWT. Immunofluorescence staining confirmed increased numbers of M2 macrophages after treatment. It was found that SWT resulted in higher number of capillaries and arterioles. Assessment of functional outcome revealed significantly improved limb perfusion in treated animals. Shock wave therapy causes increased macrophage recruitment and enhanced polarization towards reparative M2 macrophages in ischaemic muscle resulting in angiogenesis and improved limb perfusion and therefore represents a promising new treatment option for the treatment of ischaemic heart disease. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of Non-hormone Expressing Endocrine Cells in Fetal and Infant Human Pancreas.

Context: Previously, we identified chromograninA positive hormone-negative (CPHN) cells in high frequency in human fetal and neonatal pancreas, likely representing nascent endocrine precursor cells. Here, we characterize the putative endocrine fate and replicative status of these newly formed cells. Objective: To establish the replicative frequency and transcriptional identity of CPHN cells, extending our observation on CPHN cell frequency to a larger cohort of fetal and infant pancreas. Design, Setting, and Participants: 8 fetal, 19 infant autopsy pancreata were evaluated for CPHN cell frequency; 12 fetal, 24 infant/child pancreata were evaluated for CPHN replication and identity. Results: CPHN cell frequency decreased 84% (islets) and 42% (clusters) from fetal to infant life. Unlike the beta-cells at this stage, CPHN cells were rarely observed to replicate (0.2 ± 0.1 vs. 4.7 ± 1.0%, CPHN vs. islet hormone positive cell replication, p < 0.001), indicated by the lack of Ki67 expression in CPHN cells whether located in the islets or in small clusters, and with no detectable difference between fetal and infant groups. While the majority of CPHN cells express (in overall compartments of pancreas) the pan-endocrine transcription factor NKX2.2 and beta-cell specific NKX6.1 in comparable frequency in fetal and infant/child cases (81.9 ± 6.3 vs. 82.8 ± 3.8% NKX6.1+-CPHN cells of total CPHN cells, fetal vs. infant/child, p = 0.9; 88.0 ± 4.7 vs. 82.1 ± 5.3% NKX2.2+-CPHN cells of total CPHN cells, fetal vs. infant/child, p = 0.4), the frequency of clustered CPHN cells expressing NKX6.1 or NKX2.2 is lower in infant/child vs. fetal cases (1.2 ± 0.3 vs. 16.7 ± 4.7 clustered NKX6.1+-CPHN cells/mm2, infant/child vs. fetal, p < 0.01; 2.7 ± 1.0 vs. 16.0 ± 4.0 clustered NKX2.2+-CPHN cells/mm2, infant/child vs. fetal, p < 0.01). Conclusions: The frequency of CPHN cells declines steeply from fetal to infant life, presumably as they differentiate to hormone-expressing cells. CPHN cells represent a non-replicative pool of endocrine precursor cells, a proportion of which are likely fated to become beta-cells. Precis : CPHN cell frequency declines steeply from fetal to infant life, as they mature to hormone expression. CPHN cells represent a non-replicative pool of endocrine precursor cells, a proportion of which are likely fated to become beta-cells.