CT-based and morphological comparison of glenoid inclination and version angles and mineralisation distribution in human body donors.

BACKGROUND: For optimal prosthetic anchoring in omarthritis surgery, a differentiated knowledge on the mineralisation distribution of the glenoid is important. However, database on the mineralisation of diseased joints and potential relations with glenoid angles is limited. METHODS: Shoulder specimens from ten female and nine male body donors with an average age of 81.5 years were investigated. Using 3D-CT-multiplanar reconstruction, glenoid inclination and retroversion angles were measured, and osteoarthritis signs graded. Computed Tomography-Osteoabsorptiometry (CT-OAM) is an established method to determine the subchondral bone plate mineralisation, which has been demonstrated to serve as marker for the long-term loading history of joints. Based on mineralisation distribution mappings of healthy shoulder specimens, physiological and different CT-OAM patterns were compared with glenoid angles. RESULTS: Osteoarthritis grades were 0-I in 52.6% of the 3D-CT-scans, grades II-III in 34.3%, and grade IV in 13.2%, with in females twice as frequently (45%) higher grades (III, IV) than in males (22%, III). The average inclination angle was 8.4°. In glenoids with inclination ≤10°, mineralisation was predominantly centrally distributed and tended to shift more cranially when the inclination raised to > 10°. The average retroversion angle was - 5.2°. A dorsally enhanced mineralisation distribution was found in glenoids with versions from - 15.9° to + 1.7°. A predominantly centrally distributed mineralisation was accompanied by a narrower range of retroversion angles between - 10° to - 0.4°. CONCLUSIONS: This study is one of the first to combine CT-based analyses of glenoid angles and mineralisation distribution in an elderly population. The data set is limited to 19 individuals, however, indicates that superior inclination between 0° and 10°-15°, and dorsal version ranging between - 9° to - 3° may be predominantly associated with anterior and central mineralisation patterns previously classified as physiological for the shoulder joint. The current basic research findings may serve as basic data set for future studies addressing the glenoid geometry for treatment planning in omarthritis.

Anatomy of the female pelvic nerves: a macroscopic study of the hypogastric plexus and their relations and variations.

The autonomic nerves of the lesser pelvis are particularly prone to iatrogenic lesions due to their exposed position during manifold surgical interventions. Nevertheless, the cause of rectal and urinary incontinence or sexual dysfunctions, for example after rectal cancer resection or hysterectomy, remains largely understudied, particularly with regard to the female pelvic autonomic plexuses. This study focused on the macroscopic description of the superior hypogastric plexus, hypogastric nerves, inferior hypogastric plexus, the parasympathetic pelvic splanchnic nerves and the sympathetic fibres. Their arrangement is described in relation to commonly used surgical landmarks such as the sacral promontory, ureters, uterosacral ligaments, uterine and rectal blood vessels. Thirty-one embalmed female pelvises from 20 formalin-fixed and 11 Thiel-fixed cadavers were prepared. In all cases explored, the superior hypogastric plexus was situated anterior to the bifurcation of the abdominal aorta. In 60% of specimens, it reached the sacral promontory, whereas in 40% of specimens, it continued across the pelvic brim until S1. In about 25% of the subjects, we detected an accessory hypogastric nerve, which has not been systematically described so far. It originated medially from the inferior margin of the superior hypogastric plexus and continued medially into the presacral space. The existence of an accessory hypogastric nerve was confirmed during laparoscopy and by histological examination. The inferior hypogastric plexuses formed fan-shaped plexiform structures at the end of both hypogastric nerves, exactly at the junction of the ureter and the posterior wall of the uterine artery at the uterosacral ligament. In addition to the pelvic splanchnic nerves from S2-S4, which joined the inferior hypogastric plexus, 18% of the specimens in the present study revealed an additional pelvic splanchnic nerve originating from the S1 sacral root. In general, form, breadth and alignment of the autonomic nerves displayed large individual variations, which could also have a clinical impact on the postoperative function of the pelvic organs. The study serves as a basis for future investigations on the autonomic innervation of the female pelvic organs.

High metabolic activity of tissue-nonspecific alkaline phosphatase not only in young but also in adult bone as demonstrated using a new histochemical detection protocol.

Tissue-nonspecific alkaline phosphatase (TNAP) is playing a key role in bone calcification, as has been demonstrated in different mammalian species including human and rodents. However, to investigate age-related changes during life history, histochemical demonstration of TNAP is severely hampered, particularly in the elderly, by technical difficulties associated with sectioning calcified tissue. Sufficient fixation must precede decalcification since poorly fixed bone tissue is exposed to the deleterious effects of decalcification reagents. In order to find a method that would allow cryosectioning of bone without loss of TNAP activity, we assessed the efficacy of different fixation reagents regarding the effects on structural integrity and TNAP activity using liver and osseous tissue from younger and older horses. The results of this study reveal that glyoxal-based fixatives sufficiently preserved bone tissue for successful cryosectioning without compromising TNAP activity. The method described combines the demonstration of TNAP activity with optimal preservation of tissue morphology in osseous tissue of younger and even of older mammals. As a model species, we selected horse bones in light of potentially higher similarities to ageing history and lifelong locomotion in humans as compared to other, mostly smaller, experimental model species with a much shorter life span and artificial locomotive activity when kept in cages. This may serve as a basis for future studies addressing the impact of different life traits in iconic, domestic and companion animals, which are often patients in veterinary medicine, as well as for basic research on human physiology and pathologies of the musculoskeletal system.

Glenoid morphology in light of anatomical and reverse total shoulder arthroplasty: a dissection- and 3D-CT-based study in male and female body donors.

BACKGROUND: Placement of the glenoid baseplate is of paramount importance for the outcome of anatomical and reverse total shoulder arthroplasty. However, the database around glenoid size is poor, particularly regarding small scapulae, for example, in women and smaller individuals, and is derived from different methodological approaches. In this multimodality cadaver study, we systematically examined the glenoid using morphological and 3D-CT measurements. METHODS: Measurements of the glenoid and drill hole tunnel length for superior baseplate screw placement were recorded to define size of the glenoid and the distance to the scapular notch on cadaveric specimens. Glenoid angles were determined on both, 3D-CT-scans of the thoraxes using the Friedman method and on subsequently isolated scapulae from 18 male and female donors (average 84 years, range 60-98 years). RESULTS: Mean glenoid height was 36.6 mm ± 3.6, and width 27.8 mm ± 3.1 with a significant sex dimorphism (p ≤ 0.001): in males, glenoid height 39.5 mm ± 3.5, and width 30.3 mm ± 3.3, and in females, glenoid height 34.8 mm ± 2.2, and width 26.2 mm ± 1.6. The average distance from the superior screw entry to its exit in the scapular notch measured by calliper was 27.2 mm ± 6.0 with a sex difference: in males, 29.4 mm ± 5.7, and in females, 25.8 mm ± 5.9 mm with a minimum recorded distance of 15 mm. Measured by CT, the mean inclination angle for male and female donors combined was 13.0° ± 7.0, and the ante-/retroversion angle -1.0° ± 4.0°. CONCLUSION: This study is one of the first to combine dissection, including drill holes, with anatomical measurements and radiological data. In some women and smaller individuals, smaller baseplates should be selected. The published safe zone of 20 mm is generally feasible for superior screw placement, however, in small patients this distance may be substantially shorter than expected and start as of 13 and 15 mm, respectively. No correlation between glenoid height or width with the length of our drilling canal towards the scapular notch was found. Preoperative CT-based treatment planning to determine version and inclination angles is recommended.

Insulin-like growth factor I is expressed in classical and nodular lymphocyte-predominant Hodgkin's lymphoma tumour and microenvironmental cells.

Hodgkin's lymphoma (HL) is among the most frequent nodal lymphomas in the Western world and is classified into two disease entities: nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL) and classical Hodgkin's lymphoma (cHL, 95% of all HL). HL lesions are characterised by a minority of clonal neoplastic cells, namely Hodgkin and Reed-Sternberg (HRS) cells and their variants in cHL and lymphocyte-predominant (LP) cells in NLPHL, both occurring within a microenvironment of, for example, reactive T and B cells, macrophages and granulocytes that are assumed to support the proliferation and maintenance of neoplastic cells through cytokines, chemokines and growth factors. Insulin-like growth factor I (IGF-I) is an important growth factor involved in proliferation, differentiation, apoptosis and cell survival of numerous (including immune) tissues and probably has a role in tumour pathogenesis and maintenance. Although HL is characterised by disturbed cell differentiation and apoptosis mechanisms, with the involvement of the IGF-I receptor (IGF-1R), the distinct location of IGF-I in HL has not yet been defined. We localise IGF-I by double-immunofluorescence in frequent neoplastic cells of all cHL and NLPHL cases investigated. Additionally, IGF-I immunoreactivity is detected in high endothelial venules and various immune cells within the surrounding tissue of cHL including neutrophils and macrophages. IGF-1R immunoreactivity of variable intensity is found in HRS cells and high endothelial venules within the microenvironment in cHL. We assume that autocrine and paracrine IGF-I plays an anti-apoptotic role in tumour pathogenesis and in shaping the tumour microenvironment.

Developmental oestrogen exposure differentially modulates IGF-I and TNF-α expression levels in immune organs of Yersinia ruckeri-challenged young adult rainbow trout (Oncorhynchus mykiss).

Intensified aquaculture has strong impact on fish health by stress and infectious diseases and has stimulated the interest in the orchestration of cytokines and growth factors, particularly their influence by environmental factors, however, only scarce data are available on the GH/IGF-system, central physiological system for development and tissue shaping. Most recently, the capability of the host to cope with tissue damage has been postulated as critical for survival. Thus, the present study assessed the combined impacts of estrogens and bacterial infection on the insulin-like growth factors (IGF) and tumor-necrosis factor (TNF)-α. Juvenile rainbow trout were exposed to 2 different concentrations of 17ß-estradiol (E2) and infected with Yersinia ruckeri. Gene expressions of IGF-I, IGF-II and TNF-α were measured in liver, head kidney and spleen and all 4 estrogen receptors (ERα1, ERα2, ERß1 and ERß2) known in rainbow trout were measured in liver. After 5 weeks of E2 treatment, hepatic up-regulation of ERα1 and ERα2, but down-regulation of ERß1 and ERß2 were observed in those groups receiving E2-enriched food. In liver, the results further indicate a suppressive effect of Yersinia-infection regardless of E2-treatment on day 3, but not of E2-treatment on IGF-I whilst TNF-α gene expression was not influenced by Yersinia-infection but was reduced after 5 weeks of E2-treatment. In spleen, the results show a stimulatory effect of Yersinia-infection, but not of E2-treatment on both, IGF-I and TNF-α gene expressions. In head kidney, E2 strongly suppressed both, IGF-I and TNF-α. To summarise, the treatment effects were tissue- and treatment-specific and point to a relevant role of IGF-I in infection.

Oestrogen and insulin-like growth factors during the reproduction and growth of the tilapia Oreochromis niloticus and their interactions.

Oestrogens and insulin-like growth factors (Igfs) play both a central role in the regulation of reproduction and growth and can interact especially in species showing a clear-cut sex-linked growth dimorphism (SGD) like in tilapia. Aromatase is essential in ovarian differentiation and oogenesis since it controls oestrogen synthesis. During tilapia sex differentiation, aromatase cyp19a1a expression increases from 9 days post-fertilization (dpf), resulting in high oestradiol level. High temperature, exogenous androgens or aromatase inhibitors override genetic sex differentiation inducing testes development through the suppression of cyp19a1a gene expression and aromatase activity. Supplementation with 17ß-oestradiol (E2) of gonadectomized juveniles induced a sustained and higher E2 plasma level than in intact or gonadectomized controls and both sexes showed reduced growth. Juvenile and mature females treated with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione had 19% lower E2 plasma level compared to controls and they showed a 32% increased growth after 28 days of treatment. Altogether, these data suggest that E2 inhibits female growth leading to the SGD. Regarding Igf-1, mRNA and peptide appeared in liver at ∼ 4 dpf and then in organs involved in growth and metabolism, indicating a role in early growth, metabolism and organogenesis. Gonad igf-1 showed an early expression and the peptide could be detected at ∼ 7 dpf in somatic cells. It appeared in germ cells at the onset of ovarian (29 dpf) and testicular (52 dpf) meiosis. In testis, Igf-1 together with steroids may regulate spermatogenesis whereas in ovary it participates in steroidogenesis regulation. Igf-1 and Igf-2 promote proliferation of follicular cells and oocyte maturation. Igf-3 expression is gonad specific and localized in the ovarian granulosa or testicular interstitial cells. In developing gonads igf-3 is up-regulated in males but down-regulated in females. In contrast, bream Gh injections increased igf-1 mRNA in male and female liver and ovaries but gonadal igf-3 was not affected. Thus, local Igf-1 and Igf-2 may play crucial roles in the formation, development and function of gonads while Igf-3 depending on the species is involved in male and female reproduction. Furthermore, precocious ethynylestradiol (EE) exposure induced lasting effects on growth, through pituitary gh inhibition, local suppression of igf-1 expression and in testis only down-regulation of igf-3 mRNA. In conclusion, SGD in tilapia may be driven through an inhibitory effect due to E2 synthesis in female and involving Igfs regulation.

Body painting, ultrasound, clinical examination, and peer-teaching: A student-centered approach to enhance musculoskeletal anatomy learning.

The presented course, established 2016 as a compulsory elective for 22nd-year bachelor medical students, aimed to enhance deep learning of upper and lower limb anatomy from a clinical perspective by a maximum of student-centered activities combining hands-on skills training with team-learning. Three cohorts (in total 60 students) participated in this study. Students rotated through body painting, ultrasound, and clinical investigation supervised by faculty or an experienced clinician. Teams of 3-4 students prepared presentations on clinical anatomy and pathological conditions, which by teacher- and peer assessments on average achieved >85% (mean 17.8/20 points ± 1.06). After each activity session, the students reported their learning experience through a reflective diary. Fifty students (83%) evaluated the course by a voluntary anonymous questionnaire combining Likert-type scale and free-text questions to assess, predominantly, perception of course activities and their perceived influence on learning anatomy. Journal reports and questionnaires revealed that the students highly valued the course, and 92% (29 females, 17 males) rated group work satisfying or well-perceived. The highest appreciation achieved ultrasound followed by clinical examination and body painting, which one third proposed to integrate into the regular dissection course. All students recommended the course to their younger peers. This course was feasible to integrate in the pre-existing curriculum. Limiting factors to offer this elective course to more students are availability of clinical teachers, technical equipment, and education rooms. Being student-directed tasks, body painting and reflective diary-writing would be feasible to implement without additional faculty, which we recommend to educators for student engagement activation.

Morphological changes in striated muscle fibres caused by components of the Thiel embalming method.

BACKGROUND: Thiel-fixed body donors are highly valued for surgical training courses. The pronounced flexibility of Thiel-fixed tissue has been postulated to be caused by histologically visible fragmentation of striated muscle. The aim of this study was to analyse whether a specific ingredient, pH, decay, or autolysis could cause this fragmentation in order to modulate the Thiel solution to adapt specimen flexibility specifically to the needs of different courses. MATERIALS AND METHODS: Striated muscle of the mouse was fixed for different time periods in formalin, Thiel solution, and its individual ingredients, and analysed by light microscopy. Further, pH-values of Thiel solution and its ingredients were measured. In addition, unfixed muscle tissue was histologically analysed including Gram staining to investigate a relationship between autolysis, decomposition, and fragmentation. RESULTS: Muscle fixed with Thiel solution for 3 months was slightly more fragmentated than muscle fixed for 1 day. Fragmentation was more pronounced after 1 year of immersion. Three individual salt ingredients showed slight fragmentation. Decay and autolysis had no effect on fragmentation, which occurred regardless of the pH of all solutions. CONCLUSIONS: Fragmentation of Thiel-fixed muscle is dependent on fixation time and most likely occurs due to salts present in the Thiel solution. Adjustment of the salt composition in the Thiel solution with verification of the influence on the fixation effect, fragmentation and flexibility of the cadavers could be performed in further studies.

Correlation of CT-based bone mineralization with drilling-force measurements in anatomical specimens is suitable to investigate planning of trans-pedicular spine interventions.

This interdisciplinary study examined the relationship between bone density and drilling forces required during trans-pedicular access to the vertebra using fresh-frozen thoraco-lumbar vertebrae from two female body donors (A, B). Before and after biomechanical examination, samples underwent high-resolution CT-quantification of total bone density followed by software-based evaluation and processing. CT density measurements (n = 4818) were calculated as gray values (GV), which were highest in T12 for both subjects (GVmaxA = 3483.24, GVmaxB = 3160.33). Trans-pedicular drilling forces F (Newton N) were highest in L3 (FmaxB = 5.67 N) and L4 (FmaxA = 5.65 N). In 12 out of 13 specimens, GVs significantly (p < 0.001) correlated with force measurements. Among these, Spearman correlations r were poor in two lumbar vertebrae, fair in five specimens, and moderately strong in another five specimens, and highest for T11 (rA = 0.721) and L5 (rB = 0.690). Our results indicate that CT-based analysis of vertebral bone density acquired in anatomical specimens is a promising approach to predict the drilling force appearance as surrogate parameter of its biomechanical properties by e.g., linear regression analysis. The study may be of value as basis for biomechanical investigations to improve planning of the optimal trajectory and to define safety margins for drilling forces during robotic-assisted trans-pedicular interventions on the spine in the future.

The legal and ethical framework governing body donation in Europe - 2nd update on current practice.

BACKGROUND: In 2008, members of the TEPARG provided first insights into the legal and ethical framework governing body donation in Europe. In 2012, a first update followed. This paper is now the second update on this topic and tries to extend the available information to many more European countries. METHODS: For this second update, we have asked authors from all European countries to contribute their national perspectives. By this enquiry, we got many contributions compiled in this paper. When we did not get a personal contribution, one of us (EB) searched the internet for relevant information. RESULTS: Perspectives on the legal and ethical framework governing body donation in Europe. CONCLUSIONS: We still see that a clear and rigorous legal framework is still unavailable in several countries. We found national regulations in 18 out of 39 countries; two others have at least federal laws. Several countries accept not only donated bodies but also utilise unclaimed bodies. These findings can guide policymakers in reviewing and updating existing laws and regulations related to body donation and anatomical studies.

The effect of experimentally induced chronic hyperglycaemia on serum and pancreatic insulin, pancreatic islet IGF-I and plasma and urinary ketones in the domestic cat (Felis felis).

Like in humans, diabetes mellitus is on the rise in cats. Feline diabetes is a suitable model for human type-2 diabetes. We investigated magnitude and timing of insulin suppression with induced hyperglycaemia and its relationship to plasma and urinary ketones and to pancreatic islet insulin. IGF-I is under discussion as a protective mechanism but little is known about its role in diabetes in general and its distinct localisation in feline pancreatic islets in particular. Thirteen healthy, adult cats were allocated to 2 groups and infused with glucose to maintain their blood glucose at a high or moderate concentration for 42 days resulting in insulin secretion suppression. After initial increase, insulin levels declined to baseline but were still detectable in the blood at a very low level after 6 weeks of glucose infusion and then increased after a 3 week recovery period. While IGF-I in healthy cats was primarily located in glucagon cells, in hyperglycaemia-challenge IGF-I was pronounced in the ß-cells 3 weeks after ceasation of infusion. Six/8 cats developing glucose toxicity became ketonuric after 3-4 weeks. Gross lipaemia occurred approx 1 week prior to ketonuria. Ketonuric cats required 1-2 weeks of insulin therapy after-infusion until ß-cell recovery. In conclusion, ketosis and hyperlipidaemia are likely to occur in diabetic cats with glucose at 30 mmol/L, especially after ≥2 weeks. Three weeks after ceasation of infusions, clinical and morphological recovery occurred. We propose a local protective effect of IGF-I to support survival and insulin production in the hyperglycaemic state and recovery period.

Effects of seawater and freshwater challenges on the Gh/Igf system in the saline-tolerant blackchin tilapia (Sarotherodon melanotheron).

Prolactin (Prl) and growth hormone (Gh) as well as insulin-like growth factor 1 (Igf1) are involved in the physiological adaptation of fish to varying salinities. The Igfs have been also ascribed other physiological roles during development, growth, reproduction and immune regulation. However, the main emphasis in the investigation of osmoregulatory responses has been the endocrine, liver-derived Igf1 route and local regulation within the liver and osmoregulatory organs. Few studies have focused on the impact of salinity alterations on the Gh/Igf-system within the neuroendocrine and immune systems and particularly in a salinity-tolerant species, such as the blackchin tilapia Sarotherodon melanotheron. This species is tolerant to hypersalinity and saline variations, but it is confronted by severe climate changes in the Saloum inverse estuary. Here we investigated bidirectional effects of increased salinity followed by its decrease on the gene regulation of prl, gh, igf1, igf2, Gh receptor and the tumor-necrosis factor a. A mixed population of sexually mature 14-month old blackchin tilapia adapted to freshwater were first exposed to seawater for one week and then to fresh water for another week. Brain, pituitary, head kidney and spleen were excised at 4 h, 1, 2, 3 and 7 days after both exposures and revealed differential expression patterns. This investigation should give us a better understanding of the role of the Gh/Igf system within the neuroendocrine and immune organs and the impact of bidirectional saline challenges on fish osmoregulation in non-osmoregulatory organs, notably the complex orchestration of growth factors and cytokines.

Growth hormone (GH) treatment acts on the endocrine and autocrine/paracrine GH/IGF-axis and on TNF-α expression in bony fish pituitary and immune organs.

There exist indications that the growth hormone (GH)/insulin-like growth factor (IGF) axis may play a role in fish immune regulation, and that interactions occur via tumour necrosis factor (TNF)-α at least in mammals, but no systematic data exist on potential changes in GH, IGF-I, IGF-II, GH receptor (GHR) and TNF-α expression after GH treatment. Thus, we investigated in the Nile tilapia the influence of GH injections by real-time qPCR at different levels of the GH/IGF-axis (brain, pituitary, peripheral organs) with special emphasis on the immune organs head kidney and spleen. Endocrine IGF-I served as positive control for GH treatment efficiency. Basal TNF-α gene expression was detected in all organs investigated with the expression being most pronounced in brain. Two consecutive intraperitoneal injections of bream GH elevated liver IGF-I mRNA and plasma IGF-I concentration. Also liver IGF-II mRNA and TNF-α were increased while the GHR was downregulated. In brain, no change occurred in the expression levels of all genes investigated. GH gene expression was exclusively detected in the pituitary where the GH injections elevated both GH and IGF-I gene expression. In the head kidney, GH upregulated IGF-I mRNA to an even higher extent than liver IGF-I while IGF-II and GHR gene expressions were not affected. Also in the spleen, no change occurred in GHR mRNA, however, IGF-I and IGF-II mRNAs were increased. In correlation, in situ hybridisation showed a markedly higher amount of IGF-I mRNA in head kidney and spleen after GH injection. In both immune tissues, TNF-α gene expression showed a trend to decrease after GH treatment. The stimulation of IGF-I and also partially of IGF-II expression in the fish immune organs by GH indicates a local role of the IGFs in immune organ regulation while the differential changes in TNF-α support the in mammals postulated interactions with the GH/IGF-axis which demand for further investigations.

CD40 Agonist Monoclonal Antibody-Mediated Hepatitis in TNF-Receptor 1 Gene Knockout Mice.

Tumor necrosis factor-alpha (TNF-α) plays an important role in liver inflammation. CD40-CD40 ligand (CD40-CD40L) is a key receptor-ligand signaling pair involved in the adaptive immune response and pathogenesis of autoimmune diseases. In mice, CD40 activation leads to sickness behavior syndrome (SBS) comprising weight loss, sleep disruption and depression, which can be blocked by administration of the TNF-inhibitor etanercept. In the present study, we assessed the extent of hepatic inflammation in mice devoid of the TNF-receptor 1 (TNFR1)-mediated signaling pathway. The TNFR1-depleted (TNFR1-/-) adult mice and their wild type littermates were given a single intra-peritoneal injection of CD40 agonist monoclonal antibody (mAb) or rat IgG2a isotope control. As described previously, TNFR1-/- mice were protected from SBS upon CD40 mAb treatment. Cd40, tnf and tnfr1 mRNA and Tnf-α peptide were increased in the liver of CD40 mAb-stimulated wild type mice. Serum alanine aminotransferase was elevated in both CD40-activated wild type and TNFR1-/- mice. TNFR1-/- mice showed much less intra-parenchymal infiltrates, hepatocellular necrosis, and perivascular clusters upon CD40 mAb activation than their wild type littermates. A gene expression microarray detected increased activity of metabolic and detoxification pathways and decreased activity of inflammatory pathways. We conclude that immune activation and development of liver inflammation in CD40L interactions depend on TNFR1-mediated signaling pathways and are counteracted by alterations in metabolic pathways.

Micro-CT imaging of Thiel-embalmed and iodine-stained human temporal bone for 3D modeling.

INTRODUCTION: This pilot study explores whether a human Thiel-embalmed temporal bone is suitable for generating an accurate and complete data set with micro-computed tomography (micro-CT) and whether solid iodine-staining improves visualization and facilitates segmentation of middle ear structures. METHODS: A temporal bone was used to verify the accuracy of the imaging by first digitally measuring the stapes on the tomography images and then physically under the microscope after removal from the temporal bone. All measurements were compared with literature values. The contralateral temporal bone was used to evaluate segmentation and three-dimensional (3D) modeling after iodine staining and micro-CT scanning. RESULTS: The digital and physical stapes measurements differed by 0.01-0.17 mm or 1-19%, respectively, but correlated well with the literature values. Soft tissue structures were visible in the unstained scan. However, iodine staining increased the contrast-to-noise ratio by a factor of 3.7 on average. The 3D model depicts all ossicles and soft tissue structures in detail, including the chorda tympani, which was not visible in the unstained scan. CONCLUSIONS: Micro-CT imaging of a Thiel-embalmed temporal bone accurately represented the entire anatomy. Iodine staining considerably increased the contrast of soft tissues, simplified segmentation and enabled detailed 3D modeling of the middle ear.

Distinct organ-specific up- and down-regulation of IGF-I and IGF-II mRNA in various organs of a GH-overexpressing transgenic Nile tilapia.

Several lines of GH-overexpressing fish have been produced and characterized concerning organ integrity, growth, fertility and health but few and contradictory data are available on IGF-I that mediates most effects of GH. Furthermore, nothing is known on IGF-II. Thus, the expression of both IGFs in liver and various extrahepatic sites of adult transgenic (GH-overexpressing) tilapia and age-matched wild-type fish was determined by real-time PCR. Both IGF-I and IGF-II mRNA were found in all organs investigated and were increased in gills, kidney, intestine, heart, testes, skeletal muscle and brain of the transgenics (IGF-I: 1.4-4-fold; IGF-II: 1.7-4.2-fold). Except for liver, brain and testis the increase in IGF-I mRNA was higher than that in IGF-II mRNA. In pituitary, no significant change in IGF-I or IGF-II mRNA was detected. In spleen, however, IGF-I and IGF-II mRNA were both decreased in the transgenics, IGF-I mRNA even by the 19-fold. In agreement, in situ hybridisation revealed a largely reduced number of IGF-I mRNA-containing leukocytes and macrophages when compared to wild-type. These observations may contribute to better understanding the reported impaired health of GH-transgenic fish. Growth enhancement of the transgenics may be due to the increased expression of both IGF-I and IGF-II in extrahepatic sites. It is also reasonable that the markedly enhanced expression of liver IGF-II mRNA that may mimick an early developmental stage is a further reason for increased growth.

Insulin-like growth factor-3 (IGF-3) in male and female gonads of the tilapia: development and regulation of gene expression by growth hormone (GH) and 17alpha-ethinylestradiol (EE2).

Recently, in addition to IGF-1 and IGF-2 the existence of a third form of IGF, termed IGF-3, limited to fishes, to be present only in the gonads and encoded by a separate gene has been reported. However, no further data have been presented on IGF-3. The present study on tilapia (Oreochromis niloticus) uses quantitative real-time PCR specific for tilapia IGF-1 and IGF-3. The organ distribution of IGF-3 mRNA in adult fish and the early ontogeny of IGF-3 in male and female gonads were studied. The potential sensitivity of IGF-3 to GH was revealed by intraperitoneal injections of bream GH using IGF-1 as control gene. The effects of 17alpha-ethinylestradiol (EE2) exerted after feeding of high EE2 doses and exposure to low environmentally relevant EE2 doses on IGF-3 expression in testis and ovary during early development were determined. Low IGF-3 mRNA expression levels were detected in most organs studied, with the highest extra-gonadal amount in the pituitary. During development, the IGF-3 gene was significantly upregulated in male but downregulated in female gonad. Injections of GH elevated IGF-1 mRNA in male and female liver and ovary. IGF-3 did not respond to GH treatment neither in ovary nor in testis. Both EE2 treatments resulted in significant downregulations of IGF-3 mRNA in testis while ovarian IGF-3 mRNA did not respond. Thus, IGF-3 may be involved in reproduction of fishes most likely in the male gonad only. Whether IGF-3 also has some physiological significance in ovary or other organs should be the topic of further studies.

Insulin-like growth factor I messenger RNA and protein are expressed in the human lymph node and distinctly confined to subtypes of macrophages, antigen-presenting cells, lymphocytes and endothelial cells.

Insulin-like growth factor I (IGF-I) is a potent hormone that stimulates growth and differentiation and inhibits apoptosis in numerous tissues. Preliminary evidence suggests that IGF-I exerts differentiating, mitogenic and restoring activities in the immune system but the sites of synthesis of local IGF-I are unknown. Identification of these sites would allow the functional role of local IGF-I to be clarified. The presence of IGF-I in non-immune cells suggests that it acts as a trophic factor, while its occurrence in subtypes of lymphocytes or antigen-presenting cells indicates paracrine/autocrine direct regulatory involvement of IGF-I in the human immune response. The present study investigated the location of IGF-I messenger RNA and protein on archival human lymph node samples by in situ hybridization, immunohistochemistry and double immunofluorescence staining using an IGF-I probe and antisera specific for human IGF-I and CD3 (T lymphocytes), CD20 (B lymphocytes), CD68 (macrophages), CD21 (follicular dendritic cells), S100 (interdigitating dendritic cells) and podoplanin (fibroblastic reticular cells). Numerous cells within the B- and T-cell compartments expressed the IGF-I gene, and the majority of these cells were identified as macrophages. Solitary follicular dendritic cells exhibited IGF-I. A few T lymphocytes, and no B lymphocytes, contained IGF-I immunoreactive material. Furthermore, IGF-I immunoreactive cells outside the follicles that did not react with CD3, CD20, S100 or podoplanin markers were identified as high-endothelial venule (HEV) cells. From this we conclude that the main task of IGF-I in human non-tumoral lymph node may be autocrine and paracrine regulation of the differentiation, stimulation and survival of lymphocytes, antigen-presenting cells and macrophages and the differentiation and maintenance of HEV cells.

Challenge with 17alpha-ethinylestradiol (EE2) during early development persistently impairs growth, differentiation, and local expression of IGF-I and IGF-II in immune organs of tilapia.

The enormous expansion of world-wide aquaculture has led to increasing interest in the regulation of fish immune system. Estrogen has recently been shown to inhibit the endocrine (liver-derived) and autocrine/paracrine local insulin-like growth factor-I system in fish. In order to address the potential actions of estrogen on the IGF system in immune organs, tilapia were fed with 17alpha-ethinylestradiol (EE2)-enriched food from 10 to 40 days post fertilization (DPF) to induce functional feminization, an approach commonly used in aquaculture. EE2-treated and control fish were sampled at 75 and 165 DPF. The expression levels of ER-alpha, IGF-I, IGF-II and growth hormone receptor (GH-R) mRNA in spleen and head kidney were determined by real-time PCR and the expressing sites of IGF-I mRNA identified by in situ hybridisation. Ratios of spleen length and weight to body length and weight were determined. At 165 DPF, the length (4.9% vs. 7.6%) and weight (0.084% vs. 0.132%) ratios were significantly lowered in EE2-treated fish and number and size of the melanomacrophage centres were considerably reduced. At 75 DPF, both in spleen and head kidney of EE2-treated fish the expression levels of IGF-I and IGF-II mRNA were markedly diminished. The suppression was more pronounced for IGF-I (spleen: -12.071-fold; head kidney: -8.413-fold) than for IGF-II (spleen: -4.102-fold; head kidney: -1.342-fold). In agreement, clearly fewer leucocytes and macrophages in head kidney and spleen of EE2-treated fish contained IGF-I mRNA as shown by in situ hybridisation. ER-alpha mRNA expression in spleen was increased at 75 DPF but unchanged in head kidney. GH-R gene expression showed a mild upregulation at 165 DPF in both tissues. Thus, exposure to EE2 during early development affected distinctly the IGF system in tilapia immune organs. It led to lasting impairment of spleen growth and differentiation that can be attributed to an interaction of EE2 with IGF-I and, less pronouncedly, IGF-II. Especially, the impairment of spleen and melanomacrophage centres might interfere with the antigen presentation capacity of the immune system and, thus, alter susceptibility to infection.