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Layer II of the medial entorhinal cortex (MEC LII) contains the largest number of spatially modulated grid cells and is one of the first regions in the brain to express Alzheimer’s disease (AD)-related pathology. The most common principal cell type in MEC LII, reelin-expressing stellate cells, are grid cell candidates. Recently we found evidence that γ-aminobutyric acid (GABA)A receptor subunits show a specific distribution in MEC LII, in which GABAA α3 is selectively associated with reelin-positive neurons, with limited association with the other principal cell type, calbindin (CB)-positive pyramidal neurons. Furthermore, the expression of α3 subunit decreases in mice between P15 and P25, which coincides with the emergence of stable grid cell activity. It has been shown that the α3 subunit undergoes specific developmental changes and that it may exert pro-inflammatory actions if improperly regulated. In this review article, we evaluate the changing kinetics of α3-GABAA receptors (GABAARs). during development in relation to α3-subunit expression pattern in MEC LII and conclude that α3 could be closely related to the stabilization of grid cell activity and theta oscillations. We further conclude that dysregulated α3 may be a driving factor in early AD pathology. Copyright © 2019 Berggaard, Witter and van der Want. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms .

Glioblastomas (GBMs), a type of highly malignant brain tumour, contain various macrophages/microglia that are known as tumour‑associated macrophages (TAMs). These TAMs have various roles in tumour biology. Histopathological aspects of TAMs and associations with tumour growth assessed by magnetic resonance imaging (MRI) are poorly described. In the present study, 16 patients that had sufficient tumour tissue and histological hallmarks were examined. The tumours were classified as either slow‑ (n=7) or fast‑growing (n=9) based on the segmented tumour volumes from MRI scans taken at diagnosis and preoperatively. Using cluster of differentiation (CD)68 and ionized calcium-binding adaptor molecule 1 (Iba1) antibodies, the number, morphology, localization and distribution of TAMs in the GBM tissue were studied. TAMs were significantly more immunoreactive for anti‑Iba1 (TAMsIba1) compared with anti‑CD68 (TAMsCD68; P

GABAergic parvalbumin-expressing (PV+) interneurons provide powerful inhibitory modulation of grid cells in layer II of the medial entorhinal cortex (MEC LII). However, the molecular machinery through which PV+ cells regulate grid cell activity is poorly defined. PV+ interneurons impart inhibitory modulation primarily via GABA-A receptors (GABAARs). GABAARs are pentameric ion channels assembled from a repertoire of 19 subunits. Multiple subunit combinations result in a variety of receptor subtypes mediating functionally diverse postsynaptic inhibitory currents. Whilst the broad expression patterns of GABAAR subunits within the EC have been reported, those expressed by individual MEC LII cell types, in particular grid cells candidates, stellate and pyramidal cells, are less well described. Stellate and pyramidal cells are distinguished by their selective expression of reelin (RE+) and calbindin (CB+) respectively. Thus, the overall aim of this study was to provide a high resolution analysis of the major (α and γ) GABAAR subunits expressed in proximity to somato-dendritic PV+ boutons, on RE+ and CB+ cells, using immunohistochemistry, confocal microscopy and quantitative RT-PCR (qPCR). Clusters immunoreactive for the α1 and γ2 subunits decorated the somatic membranes of both RE+ and CB+ cells and were predominantly located in apposition to clusters immunoreactive for PV and vesicular GABA transporter (VGAT), suggesting expression in GABAergic synapses innervated by PV interneurons. Although intense α2 subunit-immunopositive clusters were evident in hippocampal fields located in close proximity to the EC, no specific signal was detected in MEC LII RE+ and CB+ profiles. Immunoreactivity for the α3 subunit was detected in all RE+ somata. In contrast, only a sub-population of CB+ cells was α3 immunopositive. These included CB-α3 cells which were both PV+ and PV−. Furthermore, α3 subunit mRNA and immunofluorescence decreased significantly between P 15 and P 25, a period implicated in the functional maturation of grid cells. Finally, α5 subunit immunoreactivity was detectable only on CB+ cells, not on RE+ cells. The present data demonstrates that physiologically distinct GABAAR subtypes are selectively expressed by CB+ and RE+ cells. This suggests that PV+ interneurons could utilize distinct postsynaptic signaling mechanisms to regulate the excitability of these different, candidate grid cell sub-populations. Copyright © 2018 Berggaard, Seifi, van der Want and Swinny. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).

Visual Abstract, Grid cells in layer II of the medial entorhinal cortex (MEC LII) generate multiple regular firing fields in response to the position and speed of an individual within the environment. They exhibit a protracted postnatal development and, in the adult, show activity differences along the dorsoventral axis (DVA). Evidence suggests parvalbumin-positive (PV+) interneurons, most of which are perisomatic-targeting cells, play a crucial role in generation of the hexagonal grid cell activity pattern. We therefore hypothesized that the development and organization of PV+ perisomatic terminals in MEC LII reflect the postnatal emergence of the hexagonal firing pattern and dorsoventral differences seen in grid cell activity. We used immuno-electron microscopy to examine the development of PV+ perisomatic terminals and their target somata within dorsal and ventral MEC LII in rats of postnatal day (P)10, P15, and P30. We demonstrate that in dorsal and ventral MEC LII, the cross-sectional area of somata and number and density of perisomatic PV+ terminals increase between P10 and P15. A simultaneous decrease was observed in cross-sectional area of PV+ terminals. Between P15 and P30, both MEC regions showed an increase in PV+ terminal size and percentage of PV+ terminals containing mitochondria, which may enable grid cell activity to emerge and stabilize. We also report that dorsal somata are larger and apposed by more PV+ terminals than ventral somata at all stages, suggesting a protracted maturation in the ventral portion and a possible gradient in soma size and PV+ basket innervation along the DVA in the adult.

During CNS development, interneuron precursors have to migrate extensively before they integrate in specific microcircuits. Known regulators of neuronal motility include classical neurotransmitters, yet the mechanisms that assure interneuron dispersal and interneuron/projection neuron matching during histogenesis remain largely elusive. We combined time-lapse video microscopy and electrophysiological analysis of the nascent cerebellum of transgenic Pax2-EGFP mice to address this issue. We found that cerebellar interneuronal precursors regularly show spontaneous postsynaptic currents, indicative of synaptic innervation, well before settling in the molecular layer. In keeping with the sensitivity of these cells to neurotransmitters, ablation of synaptic communication by blocking vesicular release in acute slices of developing cerebella slows migration. Significantly, abrogation of exocytosis primarily impedes the directional persistence of migratory interneuronal precursors. These results establish an unprecedented function of the early synaptic innervation of migrating neuronal precursors and demonstrate a role for synapses in the regulation of migration and pathfinding. © 2017. This is the authors’ accepted and refereed manuscript to the article. Locked until 14.11.2018 due to copyright restrictions.

The most common clinical tachycardia, Atrial Fibrillation (AF), is a progressive disease, caused by cardiomyocyte remodeling, which finally results in contractile dysfunction and AF persistence. Recently, we identified a protective role of heat shock proteins (HSPs), especially the small HSPB1 member, against tachycardia remodeling in experimental AF models. Our understanding of tachycardia remodeling and anti-remodeling drugs is currently hampered by the lack of suitable (genetic) manipulatable in vivo models for rapid screening of key targets in remodeling. We hypothesized that Drosophila melanogaster can be exploited to study tachycardia remodeling and protective effects of HSPs by drug treatments or by utilizing genetically manipulated small HSP-overexpressing strains. Tachypacing of Drosophila pupae resulted in gradual and significant cardiomyocyte remodeling, demonstrated by reduced contraction rate, increase in arrhythmic episodes and reduction in heart wall shortening, compared to normal paced pupae. Heat shock, or pretreatment with HSP-inducers GGA and BGP-15, resulted in endogenous HSP overexpression and protection against tachycardia remodeling. DmHSP23 overexpressing Drosophilas were protected against tachycardia remodeling, in contrast to overexpression of other small HSPs (DmHSP27, DmHSP67Bc, DmCG4461, DmCG7409, and DmCG14207). (Ultra)structural evaluation of the tachypaced heart wall revealed loss of sarcomeres and mitochondrial damage which were absent in tachypaced DmHSP23 overexpressing Drosophila. In addition. tachypacing induced a significant increase in calpain activity, which was prevented in tachypaced Drosophila overexpressing DmHSP23. Tachypacing of Drosophila resulted in cardiomyocyte remodeling, which was prevented by general HSP-inducing treatments and overexpression of a single small HSP, DmHSP23. Thus, tachypaced D. melanogaster can be used as an in vivo model system for rapid identification of novel targets to combat AF associated cardiomyocyte remodeling. (C) 2011 Elsevier Ltd. All rights reserved.

Contains fulltext : 87819.pdf (Publisher’s version ) (Closed access) Structural neuronal plasticity is present in the nucleus para-retroambiguus (NPRA) and the commissural nucleus of the solitary tract/A2 group (NTScom/A2) in female hamsters. Both brainstem nuclei play a role in estrous cycle related autonomic adaptations. We investigated how aging affects the capillary condition in these adaptive brainstem regions. Senescent female hamsters (+/-95 weeks) were tested weekly for their 4-day estrous cycle. Subsequently morphological changes of NPRA and NTScom/A2 were compared with those of young (+/-20 weeks) females in an ultrastructural study. The medial tegmental field served as control area. In 841 capillaries (n=319 capillaries, young females (N=3); n=522 capillaries, aged females (N=4)) vascular aberrations were classified into 3 categories: endothelial and tight junction, basement membrane and pericyte aberrations. In old animals, capillaries showed marked endothelial changes, disrupted tight junctions, and thickening and splitting of basement membranes. Aberrations were found in 40-60% of all capillaries. About 70% of the pericytes contained degenerative inclusions. Despite this generalized vascular degeneration, the reproductive cycle of female hamsters was unaffected by vascular senescence. Perivascular fibrosis as reported in aging rats was never observed, which suggests the existence of species differences. 01 augustus 2010

In vivo, homozygous staggerer (Rora(sg/sg)) Purkinje cells (PCs) remain in an early stage of development with rudimentary spineless dendrites, associated with a lack of parallel fiber (PF) input and the persistence of multiple climbing fibers (CFs). In this immunocytochemical study we used cerebellar organotypic cultures to monitor the development of Rora(sg/sg) PF-PC synapses in the absence of CF innervation. Ex vivo the vesicular glutamate transporters VGluT1 and VGluT2 reactivity was preferentially localized around the Rora(sg/sg) PC soma and proximal dendrites, which are typically CF domains. The shift from VGluT2 to VGluT1 in PF terminals during development was delayed in Rora(sg/sg) slices. The postsynaptic receptors mGluR1 and GluR52 were differently distributed on Rorasglsg PCs. mGluR1 reactivity was evenly distributed in PC soma and dendrites, whereas GluRS2 reactivity, normally restricted at PF synapses, was dense in Rora(sg/sg) PC somata. The presynaptic distribution of VGluT1 and VGluT2 on Rora'glsg PCs matched the postsynaptic distribution of the glutamate receptor GluR82, but not mGluR1. J. Comp. Neurol. 512: 467-477,2009. (c) 2008 Wiley-Liss, Inc.

Hematopoietic cells have been demonstrated to survive in many nonhematopoietic tissues after transplantation. Apparent "bone marrow-derived" cerebellar Purkinje cells in fact result from fusion events and it has been suggested that fusion may be a natural physiological phenomenon to rescue dysfunctioning cells. Here, we show that fusion of transplanted bone marrow cells with resident Purkinje cells is age-dependent and is strongly enhanced when Purkinje cells are damaged by high-dose irradiation. In addition, Purkinje heterokaryons occur in increased frequencies in the cerebellum of normal, unperturbed, aged mice compared to young animals. Our data suggest that age- and/or irradiation-induced dysfunctioning of Purkinje cells in the cerebellum is required for cell fusion. (C) 2008 Elsevier B.V. All rights reserved.

The corticotropin-releasing factor (CRF) type 2 receptor has three splice variants alpha, beta, and gamma. In the rodent brain only CRF-R2 alpha is present. In the cerebellum, CRF-R2 alpha has two different isoforms: a full-length form (fl) and truncated (tr). Both forms CRF-R2 have a unique cellular distribution. During postnatal cerebellar development, the expression patterns of tr and fl isoforms are changing. This suggests that, CRF and the related peptide urocortin (UCN) could play distinct roles in the immature and adult cerebellum, acting via different receptors subtypes. This review focuses on differences in the distribution of each isoform of CRF-R2 in view of their relationship to CRF and UCN release sites and their possible functional implications. Moreover, it includes novel findings of molecular pathways activating CRF-R2 isoforms through which CRF and UCN excert their specific actions.

Understanding what are the mechanisms that strengthen, stabilize and restrict synaptic innervation is a relevant topic in glutamate receptor delta 2 (GluR delta 2)-related research. It also involves targeting and selection of afferent input during formation of the neuronal circuitry in the cerebellar cortex and its functioning. This review will focus oil the role of GluR delta 2, one of the main players in this field. Special emphasis will be placed on the processes that regulate the rapid translocation from climbing fibres to parallel fibres of GluR delta 2 and the role of GluR delta 2 in the reduction of supernumerary climbing fibre contacts on a single Purkinje cell. Furthermore, GluR delta 2 knockout mice show ataxia and impaired motor coordination, suggesting that the presence of GluR delta 2 plays an important role in controlling cerebellar functioning. (c) 2007 Elsevier Ltd. All rights reserved.

The present study describes for the first time the neural expression and distribution of UGS148, a protein encoded by the RIKEN cDNA63330403K07 gene that has been shown to be prominently and characteristically expressed in neural stem cells (NSCs). Based on its molecular structure, UGS148 is an intracellular protein expected to be involved in intracellular sorting, trafficking, exocytosis and membrane insertion of proteins. We demonstrate that UGS148 is highly expressed in embryonic NSCs as well as, albeit at low level, in the adult neurogenic niches, the subventricular zone and the hippocampal dentate gyrus. Interestingly, the highest expression level of UGS148 in the adult mouse brain was observed specifically in the neurogenic cells lining the third ventricle, the tanycytes. Our in vitro studies show the involvement of UGS148 in the regulation of the proliferation of NSCs. (C) 2015 Elsevier Inc. All rights reserved.

Lipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitutively synthesize and secrete insulin, which is biologically functional. Insulin concentrations and release are fat mass- and LDs-dependent respectively. Fat reduction mediated by bariatric surgery repairs obesity-associated T2D. The expression of genes, like PCSK1 (proinsulin conversion enzyme), GCG (Glucagon), GPLD1, CD38 and NNAT, involved in insulin regulation/release were differentially expressed in pancreas and adipose tissue (AT). INS (insulin) and GCG expression reduced in human AT-T2D as compared to AT-control, but remained unchanged in pancreas in either state. Insulin levels (mRNA/protein) were higher in AT derived from prediabetes BB rats with destructed pancreatic β-cells and controls than pancreas derived from the same rats respectively. Insulin expression in 10 human primary cell types including adipocytes and macrophages is an evidence for extrapancreatic insulin-producing cells. The data suggest a crosstalk between AT and pancreas to fine-tune energy metabolic system or may minimize the metabolic damage during diabetes. This study opens new avenues towards T2D therapy with a great impact on public health.

Corticotropin-releasing factor (CRF) and urocortin (UCN) are closely related multifunctional regulators, governing, among other processes, Purkinje cell development. Here, we investigate the effects of CRF and UCN on Purkinje cells in organotypic slices. We show that both peptides upregulate delta 2 ionotropic glutamate receptor gene expression, and increase the abundance of the receptor in the postsynaptic density. However, only UCN treatment results in increased delta 2 protein level per Purkinje cell, implying the existence of posttranscriptional regulation of GluR delta 2 mRNA. CRF, in contrast, reduces the number of delta 2-positive dendritic shafts per cell, implying that the increase of GluR delta 2 in remaining synapses may be mainly due to its retargeting. We further observed different patterns of GluR delta 2 distribution in the zone of postsynaptic density upon CRF and UCN treatment. CRIT treatment results in a clustered distribution of GluR delta 2 along the postsynaptic density, whereas UCN treatment provides a linear distribution. (c) 2005 Elsevier Inc. All rights reserved.

In this study the effects of hypergravity were analyzed on cerebellar Purkinje cells during early development in rats. The cerebellum is a key structure in the control and the adaptation of posture and anti-gravity activities. This holds particularly when external conditions are modified. Three groups of rats were conceived, born and reared in hypergravity (2 g). At postnatal day 5 (P5), P10 or P15, they were exposed to normal gravity and at P40, the cerebella were investigated on the expression of calbindin-D28k and inositol-3-phosphate (IP3) in Purkinje cells. Control animals were bred in the same conditions but at I g. Immunoreactivity of Purkinje cells was studied in lobules III and IX of the vermis. Lobule IX of the vermis is one of the targets of primary otolithic vestibular projections, and lobule III served as a control, being much less related with vestibular inputs. The results show that hypergravity induces a decrease in calbindin and IP3 labeling in 20% of Purkinje cells of lobule IX without any change in lobule III. Animals transferred from 2g to 1 g at P5 or PI 0 showed the most pronounced effects and much less at P15. This study demonstrates that early development of the cerebellum is highly sensitive to changes in gravity. Ages until P10 are critical for the development of vestibulo-cerebellar connections, and in particularly the calcium signaling in Purkinje cells. (c) 2005 Published by Elsevier Ireland Ltd.

To investigate underlying mechanisms of thrombocytopenia in myelodysplastic syndrome (MDS), radiolabeled platelet studies were performed in 30 MDS patients with platelets

To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP), ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in 78% ± 14% of ITP megakaryocytes, which could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin. Immunohistochemistry of bone marrow biopsies of ITP patients with extensive apoptosis showed an increased number of megakaryocytes with activated caspase-3 compared with normal (28% ± 4% versus 0%). No difference, however, was observed in the number of bone marrow megakaryocyte colony-forming units (ITP, 118 ± 93/105 bone marrow cells; versus controls, 128 ± 101/105 bone marrow cells; P = .7). To demonstrate that circulating antibodies might affect megakaryocytes, suspension cultures of CD34+ cells were performed with ITP or normal plasma. Morphology compatible with (para-)apoptosis could be induced in cultured megakaryocytes with ITP plasma (2 of 10 samples positive for antiplatelet autoantibodies). Finally, the plasma glycocalicin index, a parameter of platelet and megakaryocyte destruction, was increased in ITP (57 ± 70 versus 0.7 ± 0.2; P = .009) and correlated with the proportion of megakaryocytes showing (para-) apoptotic ultrastructure (P = .02; r = 0.7). In conclusion, most ITP megakaryocytes show ultrastructural features of (para-) apoptosis, probably due to action of factors present in ITP plasma.

Background: A high throughput animal model may enhance pathophysiological studies to mechanisms of in-stent restenosis (ISR). More and appropriate antibodies and transgenic and knockout strains are available in rats. Consequently, a model for ISR in the rat would be convenient for pathobiological studies. Here we present the full characteristics of a rat ISR model suitable for high throughput stent research. Methods: The abdominal aorta of rats was separated from surrounding tissue and a BeStent™ 2 or a Cypher™ sirolimus-eluting stent was locally inserted. After 1, 3, 7, 28 and 56 days, the aortas were harvested, fixed, embedded and cut. Morphometric analysis was performed and inflammation scored. Results: The neointimal area increased to a maximum after 28 days (0.55 ± 0.08 mm2). Subsequently, the neointimal area slightly decreased. The injury score and the neointimal area were linearly correlated (r = 0.85, p < 0.01). Thrombus formation was present after 1 day. Leukocyte adherence was evident after 1 day, maximal after 3 days (93 ± 21 cells/section) and decreased thereafter. The inflammation score increased after 3 days to a maximum after 7 days (1.37 ± 0.06) and declined thereafter. After 28 days the Cypher sirolimus-eluting stent decreased the stenosis in comparison to the BeStent 2 (10.2 ± 0.85 vs. 18.0 ± 2.0%, respectively, p < 0.01). Conclusions: Stent deployment in the rat abdominal aorta results in thrombus formation, inflammation and neointimal formation. Moreover, there is a linear correlation between the injury score and the neointimal area. These responses resemble ISR events as seen in other animal models. Moreover, a known anti-restenotic stent also reduces neointimal formation in this model. Rat abdominal aorta stenting is a promising animal model for ISR, it is suitable for testing commercially manufactured stents and studying the pathophysiology of ISR.

Urocortin belongs to the family of corticotropin-releasing factor (CRF)-like peptides, which play an important role in sensorimotor coordination. CRF induces locomotor activity, and urocortin has an inhibitory effect. Here, we document the regional and subcellular localization of urocortin in the developing rat cerebellum to compare it with CRF. During the first postnatal week, urocortin immunoreactivity (UCN-ir), within the white matter and cerebellar cortex, was strongest in vermal lobules I, II, IX, and X, closely followed by lobules IV, V, and VIII; lobules VI and VII showed the weakest labeling. Cortical immunoreactivity was in the form of puncta that encircled Purkinje cell somata. By postnatal day (PD) 12, UCN-ir had increased appreciably in all lobules. In Purkinje cells, labeling was spread throughout their somata and proximal dendrites. By PD 15, labeling in lobules I-TV appeared to wane, yet still prevailed in the central and posterior lobules. This anterior-to-posterior gradient persisted through to adulthood. The study shows that urocortin and CRF have similar regional distribution profiles during development, suggesting synergistic roles within the vestibulocerebellum. The onset of the adult distributional pattern of urocortin at the stage when rats are capable of fluent walking patterns further strengthens the correlation between CRF-like peptides and postural control. An important difference between urocortin and CRF is the localization of urocortin, and not CRF, within Purkinje cells, implying that urocortin probably has an additional role in modulating the signals emanating from the cerebellar cortex to the deep cerebellar nuclei. (C) 2004 Wiley-Liss, Inc.

Molecular chaperones are involved in the protection of cells against protein damage through their ability to hold, disaggregate, and refold damaged proteins or their ability to facilitate degradation of damaged proteins. Little is known about how these processes are spatially coordinated in cells. Using a heat-sensitive nuclear model protein luciferase fused to the traceable, heat-stable enhanced green fluorescent protein (N-luc-EGFP), we now show that heat inactivation and insolubilization of luciferase were associated with accumulation of N-luc-EGFP at multiple foci throughout the nucleus. Coexpression of Hsp70, one of the major mammalian chaperones, reduced the formation of these small foci during heat shock. Instead, the heat-unfolded N-luc-EGFP accumulated in large, insoluble foci. Immunofluorescence analysis revealed that these foci colocalized with the nucleoli. Time-lapse analysis demonstrated that protein translocation to the nucleolus, in contrast to the accumulation at small foci, was fully reversible upon return to the normal growth temperature. This reversibility was associated with an increase in the level of active and soluble luciferase. Expression of a carboxyl-terminal deletion mutant of Hsp70(1–543), which lacked chaperone activity, had no effect on the localization of N-luc-EGFP, which suggests that the Hsp70 chaperone activity is required for the translocation events. Our data show that Hsp70 not only is involved in holding and refolding of heat-unfolded nuclear proteins but also drives them to the nucleolus during stress. This might prevent random aggregation of thermolabile proteins within the nucleus, thereby allowing their refolding at the permissive conditions and preventing indirect damage to other nuclear components.

In the search for better anastomosing techniques, an improved vascular stapler device (VCS clip applier system(R)) has been introduced. The system uses nonpenetrating clips to approximate everted vessel walls. The objective of this study was to determine the effects of nonpenetrating vascular clips on endothelial wound healing. Aortic end-to-end anastomoses were performed in male Wistar rats. A comparison was made between clipped (n = 12) and conventional hand-sewn (n = 6) anastomoses. Patency rates were verified at different time intervals (after 1, 4, and 8 weeks), after which the anastomotic sites were removed. Morphological evaluation was carried out using scanning electron microscopy, All rats survived the procedure. Closure with clips took less time than closure with conventional sutures, with decreasing aortic clamping times for the clipped procedures during the course of the experiments. Patency rates were 100% in both the "clipped" and "sutured" groups. Microscopic examination showed favorable endothelial healing at the clipped anastomotic sites, with less inflammatory reaction at 1 week, and a more complete endothelial regeneration at 4 and 8 weeks follow-up, as compared with the sutured anastomoses. The clip applier holds the promise of a useful device in anastomosing small-caliber vessels, since clip closure takes less time than suturing, while patency rates are identical, and morphological results are favorable. Training is mandatory to obtain technical skills and to achieve optimal results. (C) 2000 Wiley-Liss, Inc.

Purpose To determine the incidence of postoperative lens epithelial cell (LEC) layer formation anterior to the Hydroview hydrogel foldable intraocular lens (IOL), the effect on vision, and the appearance under scanning electron microscopy (SEM). Setting Eye Unit of a district general hospital, Delfzijl, and the Laboratory for Cell Biology and Electron Microscopy, Faculty of Medical Sciences, University of Groningen, The Netherlands. Methods In this prospective study, 207 eyes that received a Hydroview hydrogel foldable IOL during 1996 were followed for almost 2 years (range 8 to 98 weeks). Eleven eyes had follow-up of fewer than 8 weeks and were excluded. Some eyes had a transparent to milky-white membrane anterior to the IOL in the plane of capsulorhexis. In the absence of other contributing factors, if best corrected visual acuity was worse than expected, it was assumed to be caused by the membrane. Depending on the membrane’s thickness, determined at the slitlamp, patients were offered a neodymium:YAG (Nd:YAG) laser or surgical membranectomy. If surgically excised, the layer was studied by SEM. Results Of the 196 eyes, 62 eyes (33.16%) developed a thin epithelial cell layer. The membrane appeared a mean of 38.5 weeks postoperatively (range 9.9 to 85.6 weeks). Eleven eyes (5.61%) had visual symptoms varying from low vision to hazy vision or light scatter. Visual symptoms were present even in cases in which the visual axis was not invaded by the membrane. Once this layer was removed surgically or by the Nd:YAG laser, vision improved and symptoms were relieved. Four eyes (2.04%) required a surgical membranectomy. Examination of these removed membranes by SEM showed their multilayer nature and that the LECs had differentiated into fibroblast-like cells with collagenous fibrils and extracellular matrix. Conclusions The incidence of LEC membrane formation was higher than expected and increased with time in eyes with a Hydroview IOL. The membrane caused visual symptoms by occluding the visual axis, causing striation on the membrane and the IOL to decenter, tilt, or fold. These symptoms improved after the membrane was removed.

Several findings have indicated an involvement of dopamine in panic and defensive behaviors. The dorsolateral column of the periaqueductal gray (dlPAG) is crucially involved in the expression of panic attacks in humans and defensive behaviors, also referred to as panic-like behaviors, in animals. Although the dlPAG is known to receive a specific innervation of dopaminergic fibers and abundantly expresses dopamine receptors, the origin of this dopaminergic input is largely unknown. This study aimed at mapping the dopaminergic projections to the dlPAG in order to provide further insight into the panic-like related behavior circuitry of the dlPAG. For this purpose, the retrograde tracer cholera toxin subunit b (CTb) was injected into the dlPAG of male Wistar rats and double immunofluorescence for CTb and tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine, was performed. Neurons labeled for both CTb and TH were counted in different dopaminergic cell groups. The findings indicate that the dopaminergic nerve terminals present in the dlPAG originate from multiple dopamine-containing cell groups in the hypothalamus and mesencephalon. Interestingly, the A13 cell group is the main source of dopaminergic afferents to the dlPAG and contains at least 45% of the total number of CTb/TH-positive neurons. Anterograde tracing with biotinylated dextran amine (BDA) combined with double immunofluorescence for BDA and TH confirmed the projections from the A13 cell group to the dlPAG. The remainder of the dopamine-positive terminals present in the dlPAG was found to originate from the extended A10 cell group and the A11 group. The A13 cell group is known to send dopaminergic efferents to several other brain regions implicated in defensive behavior, including the central amygdala and ventromedial hypothalamus. Therefore, although direct behavioral evidence is lacking, our finding that the A13 cell group is also the main source of dopaminergic input to the dlPAG suggests that dopamine might contribute to the regulation of dlPAG-mediated defensive behaviors. Copyright © 2013 Messanvi, Eggens-Meijer, Roozendaal and van der Want. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

The nucleus pararetroambiguus (NPRA) and the commissural nucleus of the solitary tract (NTScom) show estrogen nuclear receptor-alpha immunoreactivity (nuclear ER-alpha-IR). Both cell groups are involved in estrous cycle related adaptations. We examined in normally cycling aged hamsters the occurrence/amount/frequency of age-related degenerative changes in NPRA and NTScom during estrus and diestrus. In 2640 electron microscopy photomicrographs plasticity reflected in the ratio of axon terminal surface/dendrite surface (t/d) was morpho-metrically analyzed. Medial tegmental field (mtf, nuclear ER-alpha-IR poor), served as control. In aged animals, irrespective of nuclear ER-alpha-IR+ or nuclear ER-alpha-IR- related cell groups, extensive diffuse degenerative structural aberrations were observed. The hormonal state had a strong influence on t/d ratios in NPRA and NTScom, but not in mtf. In NPRA and NTScom, diestrous hamsters had significantly smaller t/d ratios (NPRA, 0.750 +/- 0.050; NTScom, 0.900 +/- 0.039) than the estrous hamsters (NPRA, 1.083 +/- 0.075; NTScom, 1.204 +/- 0.076). Aging affected axodendritic ratios only in mtf (p

Objective. Indoleamine-2,3-dioxygenase (IDO) suppresses the function of T-lymphocytes and is an important immune escape mechanism for cancer. Therefore, it is to be expected that IDO influences prognosis of cancer patients. This study aimed to investigate the prognostic role of IDO expression in a large cohort of endometrial carcinoma (EC) patients.Methods. A tissue microarray containing primary EC tissue of 355 patients treated in a single institution was used to evaluate IDO expression. Expression of IDO was associated with clinicopathological characteristics, survival and previously determined numbers of CD8(+) and Foxp3(+) T-lymphocytes.Results. IDOhigh expression was associated with lower numbers of intratumoral CD8(+) T-lymphocytes (p = 0.031). Next to well-known prognostic parameters, IDOhigh expression was independently associated with poor disease specific survival in the general cohort of EC patients (HR 2.62, 95% C.I. 1.48-4.66, p = 0.001) and among patients with early stage EC (HR 3.06, 95% C.I. 1.10-8.54, p = 0.032).Conclusion. Our results show that IDO expression is associated with poor survival. This provides evidence that further research into the use of IDO blocking agents in cancer treatment is valid where it might be a promising new therapeutic strategy. (c) 2012 Elsevier Inc. All rights reserved.

Lipofuscin accumulation is a characteristic feature of senescent postmitotic neuronal cells but estrogen may have protecting effects by inhibiting its formation. In the present ultrastructural study, lipofuscin accumulation was studied in 2 estrogen-alpha-receptive brainstem areas: nucleus pararetroambiguus (NPRA) and the commissural part of the solitary tract nucleus/A2 catecholaminergic group (NTScom/A2) and compared with the estrogen-insensitive medial tegmental field (mtf), in young (23 weeks) and aged (95 weeks) female hamsters. In the aged animals, extensive intracytoplasmic lipofuscin accumulation was observed. A total number of 6450 neurons were classified in 4 categories. Levels were significantly elevated in each of the brain areas studied. Lipofuscin accumulation was strongest in the mtf, less in NPRA, and remarkably less in the area of NTScom/A2. In conclusion, the observed differences in lipofuscin accumulation suggest: (1) considerable regional differences in the degree of neuronal vulnerability; and (2) a possible neuroprotective role for estrogen, because the degree of accumulation is inversely related to the density of the estrogen receptors, varying from nonreceptive (mtf) to NPRA and NTScom/A2 (most receptive). (C) 2012 Elsevier Inc. All rights reserved.

We have studied the organization of gamma-aminobutyric acid (GABA)ergic profiles in the superior colliculus of the rabbit to determine whether the synaptic types found in cat and monkey also exist in a mammalian species whose visual system has a different organization. Ultrastructure of GABAergic profiles was examined by use of a polyclonal antibody to GABA and quantitative postembedding immunocytochemistry. Three distinct types of vesicle-containing profiles were labeled by the GABA antibody in the rabbit superior colliculus. One type was a putative presynaptic dendrite (PSD profile) that received synaptic input from other profiles and contained pleomorphic synaptic vesicles scattered throughout the profile. These PSD profiles frequently received retinal input and formed dendrodendritic synapses. A second type of profile was a large caliber dendrite, often horizontal in orientation (H profile), that had one or more discrete clusters of pleomorphic synaptic vesicles at sites of synaptic contact with conventional dendrites. These H profiles received few synaptic contacts. A third profile type was a putative axon terminal (F profile) with smaller, more flattened synaptic vesicles that densely and uniformly filled the profile. Quantitative analysis of gold particle density revealed that F profiles had a significantly higher gold particle density (14.3/microns 2) than did PSD or H profiles (10.4 and 10.2/microns 2), suggesting that GABAergic profile types contain different concentrations of GABA. The vesicle density of these profile types also differed, but no obvious relationship between vesicle and particle distributions was observed. We conclude that the profiles labeled by GABA in rabbit superior colliculus are similar to those in cat and monkey and must represent a phylogenetically conserved organization common to many mammals, and that particle density analysis of postembedding immunocytochemistry can distinguish different GABAergic profile types.

A combined electrophysiological and morphological investigation of the medial terminal nucleus (MTN) in the rat was undertaken, aimed at a better understanding of the relationship between structure and function in this nucleus. The locations of upward and downward direction selective units in the MTN were documented with extracellular electrophysiological recording. By means of tracer experiments, with Phaseolus vulgaris-leucoagglutinin, biocytin, and cholera toxin subunit B-horseradish peroxidase, the internal connections of the MTN, its retinal afferents, and the projection neurons to the inferior olive were visualized. Terminals originating from the retina and from internal connections were characterized at the ultrastructural level. Their termination pattern on cells in the MTN, including identified inferior olive projection neurons, were determined. Additionally, postembedding GABA immunocytochemistry was performed to identify GABAergic elements. From reconstructions of the positions of electrophsiologically recorded units in the MTN, a local segregation between upward and downward direction selective units was revealed. Upward direction selective units were found in the dorsal part and ventromedially, whereas downward direction selective units were found ventral and laterally in the MTN. The MTN receives optic fibers via two separate routes which, based on their trajectory, presumably terminate in different parts of the MTN: the inferior fascicle of the accessory optic tract in the dorsal part, and the posterior fiber bundle of the superior fascicle in the ventral part of the MTN. A correspondence has been found between the segregation of direction selective units and the areas in the MTN where the retinal fibers from the two pathways distribute. It is, therefore, proposed that the inferior fasciculus conveys upward direction selectivity and the posterior fiber bundle downward direction selectivity, and that the two fiber bundles terminate segregated in the MTN. After anterograde tracing from the eye, retinal terminals were found evenly distributed throughout the MTN. They are characterized as GABA negative R-type terminals. After retrograde tracing from the inferior olive, identified MTN-inferior olive projection neurons were found in the dorsal MTN and medially in the ventral MTN. Their location in the MTN suggests that MTN-inferior olive projection neurons are upward direction selective. MTN-inferior olive projection neurons are large non-GABAergic cells, with a variable form. A majority of both F- and R-type terminals were found to make synaptie contacts on the dendrites of MTN cells. MTN-inferior olive projection neurons did not differ from other neurons in this respect. A reciprocal projection between the ventral and dorsal MTN was revealed by anterograde tracing. These connections make F-type terminals which contain GABA. This indicates that the dorsally located upward direction selective neurons have reciprocal inhibitory connections with the ventrally located downward direction selective neurons. The presence of inhibitory intranuclear connections can explain the noncollinearity of the preferred and nonpreferred directions of MTN units. © 1993 Wiley-Liss, Inc.

The mammalian circadian pacemaker of the suprachiasmatic nucleus (SCN) is entrained to the environmental light-dark cycle via a retinal projection, the retinohypothalamic tract (RHT). Several studies suggest that an excitatory amino acid, possibly glutamate, is involved in photic entrainment. However, it has not yet been established whether glutamate is a transmitter of the RHT itself. We have now identified terminals of the RHT in the SCN of brown Norwegian rats by intravitreous injections of horse radish peroxidase conjugated to cholera toxin. To detect glutamate immunoreactivity (IR), post-embedding immunocytochemistry was performed with polyclonal antibodies which were visualized for electron microscopy with colloidal gold particles. Retinal terminals had a significantly 82% higher glutamate-IR than their post-synaptic dendrites and a significantly 76% higher glutamate-IR than non-retinal terminals. These observations provide ultrastructural evidence that glutamate is a transmitter of the RHT.

Pantothenate kinase–associated neurodegeneration (PKAN), a progressive neurodegenerative disorder, is associated with impairment of pantothenate kinase function. Pantothenate kinase is the first enzyme required for de novo synthesis of CoA, an essential metabolic cofactor. The pathophysiology of PKAN is not understood, and there is no cure to halt or reverse the symptoms of this devastating disease. Recently, we and others presented a PKAN Drosophila model, and we demonstrated that impaired function of pantothenate kinase induces a neurodegenerative phenotype and a reduced lifespan. We have explored this Drosophila model further and have demonstrated that impairment of pantothenate kinase is associated with decreased levels of CoA, mitochondrial dysfunction, and increased protein oxidation. Furthermore, we searched for compounds that can rescue pertinent phenotypes of the Drosophila PKAN model and identified pantethine. Pantethine feeding restores CoA levels, improves mitochondrial function, rescues brain degeneration, enhances locomotor abilities, and increases lifespan. We show evidence for the presence of a de novo CoA biosynthesis pathway in which pantethine is used as a precursor compound. Importantly, this pathway is effective in the presence of disrupted pantothenate kinase function. Our data suggest that pantethine may serve as a starting point to develop a possible treatment for PKAN.

Aims We previously showed that enhanced myogenic constriction (MC) of peripheral resistance arteries involves active AT(1) receptors in chronic heart failure (CHF). Recent data suggest both transactivation of EGF receptors and caveolae-like microdomains to be implicated in the activity of AT(1) receptors. Thus, we assessed their roles in increased MC in mesenteric arteries of CHF rats.Methods and results Mate Wistar rats underwent myocardial infarction to induce CHF and were sacrificed after 12 weeks. The number of caveolae in smooth muscle cells (SMC) of mesenteric arteries of CHF rats was decreased by 43.6 +/- 4.0%, this was accompanied by increased MC, which was fully normalized to the level of sham by antagonists of the AT(1)-receptor (losartan) or EGF-receptor (AG1478). Acute disruption of caveolae in sham rats affected caveolae numbers and MC to a similar extent as CHF, however MC was only reversed by the antagonist of the EGF-receptor, but not by the AT(1)-receptor antagonist. Further, in sham rats, MC was increased by a sub-threshold concentration of angiotensin II and reversed by both AT(1)-as well as EGF-receptor inhibition. In contrast, increased MC by a sub-threshold concentration of EGF was only reversed by EGF receptor inhibition.Conclusion These findings provide the first evidence that decreased SMC caveolae numbers are involved in enhanced MC in small mesenteric arteries, by affecting AT(1)- and EGF-receptor function. This suggests a novel mechanism involved in increased peripheral resistance in CHF.

International audience; The geometric and subcellular organization of axon arbors distributes and regulates electrical signaling in neurons and networks, but the underlying mechanisms have remained elusive. In rodent cerebellar cortex, stellate interneurons elaborate characteristic axon arbors that selectively innervate Purkinje cell dendrites and likely regulate dendritic integration. We used GFP BAC transgenic reporter mice to examine the cellular processes and molecular mechanisms underlying the development of stellate cell axons and their innervation pattern. We show that stellate axons are organized and guided towards Purkinje cell dendrites by an intermediate scaffold of Bergmann glial (BG) fibers. The L1 family immunoglobulin protein Close Homologue of L1 (CHL1) is localized to apical BG fibers and stellate cells during the development of stellate axon arbors. In the absence of CHL1, stellate axons deviate from BG fibers and show aberrant branching and orientation. Furthermore, synapse formation between aberrant stellate axons and Purkinje dendrites is reduced and cannot be maintained, leading to progressive atrophy of axon terminals. These results establish BG fibers as a guiding scaffold and CHL1 a molecular signal in the organization of stellate axon arbors and in directing their dendritic innervation.

The ultrastructural organization of the pretecto-olivary projection neurons within the nucleus of the optic tract and dorsal terminal accessory optic nucleus of rabbits was studied by using anti-GABA immunolabelling and retrograde transport of WGA-HRP. GABA-like immunoreactivity was determined with a postembedding colloidal gold technique. WGA-HRP was injected in the dorsal cap of the inferior olive. The WGA-HRP-labelled neurons were incubated with gold-substituted silver peroxidase. Neurons projecting to the inferior olive had large to medium-sized cell bodies and were GABA negative. In the nucleus of the optic tract, projection neurons are found in the rostral parts, while the majority of the local GABAergic interneurons are mainly found in the caudal parts. In the dorsal terminal nucleus both types of neurons are intermingled. The projection neurons were frequently in synaptic contact by GABAergic terminals. These neurons also receive retinal afferents indicating the existence of a two-step synaptic connection from the retina to the inferior olive. It is suggested that this class of projection neurons forms the "direction-selective" neurons that can be antidromically stimulated from the inferior olive. The GABAergic terminals on the identified projection neurons are of axonal origin (F-terminals), whereas presynaptic dendrites of interneurons (P-terminals) were seldom observed to be in synaptic contact with retrogradely labelled profiles. The strong input of GABA on direction-selective neurons indicates that GABA is directly involved in modulating retinal signals to the inferior olive.

To investigate whether the type of programmed cell death of myelodysplastic erythroid cells depends on their cellular context, we performed studies on cells from patients with low-risk myelodysplastic syndromes. We compared erythroid cells (and their precursor cells) from the mononuclear cell fraction with those from the hematon fraction, which are compacted complexes of hematopoietic cells surrounded by their own micro-environment. In directly fixed materials, erythroblasts exhibited signs of autophagy with limited apoptosis (3%) based on ultrastructural characteristics and immunogold labeling for activated caspase-3. After 24 h in culture, myelodysplastic erythroblasts exhibited a significant increase in apoptosis (22 ± 7% vs. 3 ± 2%, p = 0.001). In contrast, the myelodysplastic erythroblasts from the hematon fraction did not exhibit an increased tendency toward apoptosis after culture (7 ± 3.3% vs. 1.8 ± 2.3%), which was in line with results for normal bone marrow cells. The same dependency on the micro-environment was noted for immature erythroid progenitor cells. Myelodysplastic hematons exhibited distinct numbers of erythroid burst-forming units in association with an extensive network of stromal cells, whereas small numbers of erythroid burst-forming units were generated from the myelodysplastic mononuclear cells compared with normal mononuclear cells (10.2 ± 9 vs. 162 ± 125, p0.001). Co-culture of erythroid myelodysplastic cells in the presence of growth factors (vascular endothelial growth factor, leukemia inhibitory factor) or on the MS-5 stromal layer did not restore the expansion of erythroid precursor cells. These data indicate that surviving myelodysplastic erythroid progenitors become more vulnerable to programmed cell death when they are detached from their own micro-environment.

Disease-associated misfolded proteins or proteins damaged due to cellular stress are generally disposed via the cellular protein quality-control system. However, under saturating conditions, misfolded proteins will aggregate. In higher eukaryotes, these aggregates can be transported to accumulate in aggresomes at the microtubule organizing center. The fate of cells that contain aggresomes is currently unknown. Here we report that cells that have formed aggresomes can undergo normal mitosis. As a result, the aggregated proteins are asymmetrically distributed to one of the daughter cells, leaving the other daughter free of accumulated protein damage. Using both epithelial crypts of the small intestine of patients with a protein folding disease and Drosophila melanogaster neural precursor cells as models, we found that the inheritance of protein aggregates during mitosis occurs with a fixed polarity indicative of a mechanism to preserve the long-lived progeny., Human cells containing polyglutamine damage enter mitosis and complete cytokinesis. The association of aggresomes with one centrosome means that accumulated damage is asymmetrically inherited in only one daughter cell.

Corticotropin releasing factor receptor 2 (CRF-R2) is strongly expressed in the cerebellum and plays an important role in the development of the cerebellar circuitry, particularly in the development of the dendritic trees and afferent input to Purkinje cells. However, the mechanisms responsible for the distribution and stabilization of CRF-R2 in the cerebellum are not well understood. Here, we provide the first detailed analysis of the cellular localization of the full-length form of CRF-R2 in rat cerebellum during early postnatal development. We document unique and developmentally regulated subcellular distributions of CRF-R2 in cerebellar cell types, e.g. granule cells after postnatal day 15. The presence of one or both receptor isoforms in the same cell may provide a molecular basis for distinct developmental processes. The full-length form of CRF-R2 may be involved in the regulation of the first stage of dendritic growth and at later stages in the controlling of the structural arrangement of immature cerebellar circuits and in the autoregulatory pathway of the cerebellum.

This paper describes three types of hair cell configurations with stereo- and kinocilia in the head of the hornet; these were encountered at the vertex and frons regions adjacent to the three ocelli and are assumed to be part of the hornet's gravity detecting system together with cuticular photoreceptors. The first and most common type of hair cell configuration ( type A) was a cell surrounded by a septum, having a diameter of 30-50 mu m. Aggregates of over 20 such hair cell groups together formed a larger unit, 130-300 mu m in diameter, which was also enclosed by a septum. Many of these larger round units were, in turn, arranged in either angular or leaf-like clusters. The hair cells bore numerous cilia of 4.5-6.0 mu m long, and were themselves composed of smaller sub-units of about 7-8 pm in diameter, which were not enclosed by a septum. The second type of hair cell configuration (type B) was of discrete cells with a diameter of similar to 12.5-14 mu m, located in the vicinity of the pore canal outlet of the peripheral photoreceptor. These single hair cells were either devoid of or only partially enclosed by a septum. Their cilia were 4.5-6.0 mu m long as well, but with a diameter of only 150-160 nm. On the exterior of each cilium a tubular system could be detected. Furthermore, the tips of adjacent cilia were interconnected by a kind of fibre bearing a spherical body in its middle. The third type of hair cell (type C), present in the neighbourhood of the second type of hair cell (type B), was chalice-shaped and had interconnecting fibrils comparable to those found at type B. We believe that these three types of hair cell configuration along with the ganglion cells interconnecting their bases are all components of the gravity organ of the hornet (the Ishay Organ) and together with the cuticular photoreceptors function in the navigation system of the hornet. We further conjecture that the described structures are engulfed by endolymph; produced signals by each unit are conducted by neural fibrils to the hornet's central nervous system.

Abstract 2932 Several studies have shown that hematopoietic precursor cells in low-risk myelodysplastic syndromes (MDS) undergo premature cell death. The mode of cell death is cell type specific and might include apoptosis, necrosis, and/or autophagy. Studies in MDS erythroblasts have shown enhanced apoptosis and enhanced autophagy (Houwerzijl EJ et al. Leukemia 2009), while megakaryocytes undergo a caspase-3 independent nonapoptotic cell death (Houwerzijl EJ et al. Blood 2005). Increasing data suggest that in MDS the bone marrow microenvironment is also affected. Some stromal cells, including mesenchymal and circulating endothelial cells, can carry similar chromosomal abnormalities as the neoplastic hematopoietic clone (Della Porta MG et al. Leukemia 2008, Lopez-Villar O et al. Leukemia 2009). In addition, recent data show that hematopoietic and endothelial cells have a common precursor, the hemangioblast (Lancrin C et al. Nature 2009). These findings raise the question whether endothelial cells in MDS also die prematurely. To define in more detail the underlying cell death pathways in MDS endothelial cells and to quantify vascularisation, immunohistochemical staining and electron microscopic analysis were performed on bone marrow samples of patients with refractory anemia (RA, n=6) and RA with ringed sideroblasts (RARS, n=6), and healthy controls (n=4). According to the MDS International Prognostic Scoring System (IPSS) the patients were categorized as low risk (n=6) and intermediate risk-1 (n=6). Immunohistochemistry of the MDS bone marrow biopsies demonstrated increased bone marrow microvessel density (MVD) determined by FVIII staining. In both RA and RARS MVD was increased compared to normal (number of vessels: 4 ± 1.6 and 3.8 ± 1.1/powerfield respectively, versus 0.4 ± 0.5 in healthy controls, magnification × 400). The increased number of endothelial cells stained strongly positive for VEGF in both RA and RARS compared to normal bone marrow. The elevated VEGF expression of these cells might be related to a significantly enhanced expression of hypoxia inducible factor-2α (HIF-2α) compared to normal controls, which was especially found in RARS endothelial cells. In contrast, immunostaining for HIF-1α was negative in MDS patients as well as controls. To expand these results ultrastructural analysis was performed on hematons of a subgroup of these patients (n=10). Hematons are compact spherical particles which contain hematopoietic progenitor cells residing within a finely arborized stromal framework, including adipocytes, mesenchymal cells, resident macrophages and endothelial cells (Blazsek I et al. Blood 2000). Hematons can be isolated from the bone marrow aspirate light density fraction. The ultrastructural analysis revealed irregularly shaped endothelial cells without pericytes and degradation of the basal membrane. Cytoplasmic vacuolization was present in endothelial cells, especially in RARS patients. The majority of these vacuoles were double membraned, a main characteristic of autophagy. Mitochondria were normal. No features of apoptosis were found, which was confirmed by a negative immunostaining for caspase-3 and caspase-8. Microvessels were irregularly shaped and showed frequent sprouting. These results indicate that endothelial cells from low-risk MDS patients are ultrastructurally abnormal, showing features of autophagy, but no apoptosis. Autophagy in these cells may be a type of cell death or a cell-rescue reaction to nutrient- and/or growth factor depletion in the microenvironment. On the other hand, in agreement with previous studies, angiogenesis, in low-risk MDS is increased, and appears to be HIF-2α and not HIF-1α mediated. Together, these findings may suggest that endothelial cells in MDS, similar to hematopoietic cells, show a high rate of cellular proliferation, which coincides with an increased susceptibility for premature cell death, i.e. autophagy. The findings might contribute to knowledge of the defective make-up of the stem cell compartment in MDS which requires a strong interaction between hematopoietic stem cells and the microenvironment. Disclosures: No relevant conflicts of interest to declare.

The nucleus of the optic tract (NOT) and the medial terminal nucleus (MTN) are two primary visual nuclei that take part in circuits sustaining the optokinetic reflex, The morphology of rat NOT cells projecting to the inferior olive (NOT-IO neurons) and their terminal input, specifically terminals originating from the MTN, have been studied in the rat at the light and electron microscopical level. This has been done by means of combined retrograde tracing from the inferior olive and anterograde tracing from the MTN to the NOT. The area containing MTN terminal fibers and the area occupied by NOT-IO neurons has been found to match. This matched distribution provides a more detailed description of the NOT, with possible functional implications. Identified NOT-IO neurons demonstrate considerable variability in their dendritic branching pattern and have been found to include all neuronal cell types described for the NOT. The dendritic branching pattern of NOT-IO cells could be related to the orientation and distribution of the NOT's major afferent fiber systems. NOT-IO neurons receive a variable MTN and retinal input onto their somata, comparable to other cells in the NOT. With exception of the superficial part of the NOT, NOT-IO neurons with the most MTN terminals were found dorsally in areas containing large numbers of MTN terminals. In conclusion, although NOT-IO neurons are uniform with respect to their receptive field properties, they vary considerably with respect to the shape of the cell body, dendritic branching pattern, and terminal input. This means that morphological characteristics of NOT-IO neurons have no predictive value with regard to their receptive field properties. © 1992 Wiley-Liss, Inc.

Anemia in myelodysplasia (MDS) is partially ascribed to enhanced programmed cell death (PCD) of committed erythroid cells in the bone marrow compartment. Especially, enhanced apoptosis has been described. However, nonapoptotic forms of PCD have been demonstrated in MDS megakaryocytes characterized by the absence of chromatin condensation and caspase-3 and -8 activation (Houwerzijl EJ et al. Blood2005;105:3472–3479). Recent studies have indicated that besides apoptosis, necrosis and autophagic cell death can be recognized as PCD, whereby cells are capable to switch between the different types of PCD dependent on their cellular context. To define in more detail the underlying cell death pathways in MDS erythroblasts, immunohistochemical staining and ultrastructural and immunolabeling analysis were performed on bone marrow samples of low-risk MDS patients and normal controls (n=4). Immunohistochemistry of MDS bone marrow biopsies (n=23) demonstrated no positive staining of the erythroblasts for active caspase -3 and -8. To confirm these results ultrastructural analysis and immunoelectron microscopy was performed on mononuclear cells (MNC) and hematons of a subgroup of these patients (n=9). Hematons are compact hematopoietic complexes in which hematopoietic cells, including erythroblasts, are embedded in their own microenvironment. The ultrastructural analysis revealed that only a small fraction of erythroid cells of the MNC and hematon fraction of both MDS patients and healthy controls demonstrated features of apoptosis (2 ± 2% vs 0%). However, 52 ± 16% of immature and mature MDS erythroblasts contained cytoplasmic vacuoles in contrast to normal erythroblasts, in which vacuoles were only shown in the matured stage (12 ± 3%). These vacuoles were partly double-membraned and stained positive for the lysosomal marker LAMP (lysosome associated membrane protein)-2, catalase and the mitochondrial inner membrane protein (immunogold staining) underscoring the presence of autophagy of mitochondria and other cytoplasmic components. Morphometric analysis confirmed that the vacuolar surface in the cytoplasm of MDS erythroblasts was increased compared to controls (P

The hexagonal brood-rearing cells inside the nest combs of the hornet Vespa orientalis are uniform in both their architecture and orientation. We have discovered that each cell contains a minute crystal that projects down from the centre of its domed roof and has a composition typical of the magnetic mineral ilmenite1,2. These tiny crystals form a network that may act like a surveyor's spirit-level, helping the hornets to assess the symmetry and balance of the cells and the direction of gravity while they are building the comb.

Background Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis. Methods We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls. Results We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1. Conclusion The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD. © 2013 Hoffmann et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.