Toward safer thanatopraxy cares: formaldehyde-releasers use.

Human cadavers constitute very useful educational tools to teach anatomy in medical scholarship and related disciplines such as physiology, for example. However, as biological material, human body is subjected to decay. Thanatopraxy cares such as embalming have been developed to slow down and inhibit this decay, but the formula used for the preservation fluids are mainly formaldehyde (FA)-based. Very recently, other formulas were developed in order to replace FA, and to avoid its toxicity leading to important environmental and professional exposure concerns. However, these alternative FA-free fluids are still not validated or commercialized, and their efficiency is still under discussion. In this context, the use of FA-releasing substances, already used in the cosmetics industry, may offer interesting alternatives in order to reduce professional exposures to FA. Simultaneously, the preservation of the body is still guaranteed by FA generated over time from FA-releasers. The aim of this review is to revaluate the use of FA in thanatopraxy cares, to present its benefits and disadvantages, and finally to propose an alternative to reduce FA professional exposure during thanatopraxy cares thanks to FA-releasers use.

[« Living facing death' : improving Lausanne's medical students ¼ attitude toward end-of-life issues]. / Vivre face à la mort : faire prendre du recul à des étudiants de médecine lausannois.

The optional course « Living facing death ¼ is the result of an innovative educational approach jointly developed by CHUV's Palliative Care Service, Lausanne's School of Medicine, Medical Ethics Unit and the students' association « Doctors & Death ¼. It is intended for 3rd and 4th grade medical students and was inspired by previous experiences conducted at Harvard Medical School. Its primary objective is to help students to « take some distances ¼. A mixed quantitative/qualitative satisfaction questionnaire was administered to all participating students at the completion of the course. It confirms experiential knowledge is a valuable teaching tool to improve medical students' attitude toward end-of-life issues.

Le cours à option « Vivre face à la mort ¼ a été développé conjointement par le Service de soins palliatifs du CHUV, l'Ecole de médecine de Lausanne, l'Unité d'éthique et l'association d'étudiants Doctors and Death, en s'inspirant de réflexions pédagogiques menées à Harvard. Il est destiné à des étudiants de médecine de 3e et 4e années et utilise des outils pédagogiques novateurs pour leur permettre de « prendre de la distance ¼. Il est le résultat d'une démarche pédagogique structurée, présentée dans l'article. Une évaluation mixte quantitative/qualitative de la satisfaction des étudiants confirme que l'utilisation de l'experiential knowledge autorise un changement d'attitude des étudiants face aux enjeux de la finitude.

An organotypic slice culture to study the formation of calyx of Held synapses in-vitro.

The calyx of Held, a large axo-somatic relay synapse containing hundreds of presynaptic active zones, is possibly the largest nerve terminal in the mammalian CNS. Studying its initial growth in-vitro might provide insights into the specification of synaptic connection size in the developing brain. However, attempts to maintain calyces of Held in organotypic cultures have not been fruitful in past studies. Here, we describe an organotypic slice culture method in which calyces of Held form in-vitro. We made coronal brainstem slices with an optimized slice angle using newborn mice in which calyces have not yet formed; the presynaptic bushy cells were genetically labeled using the Math5 promoter. After six to nine days of culturing, we readily observed large Math5-positive nerve terminals in the medial nucleus of the trapezoid body (MNTB), but not in the neighboring lateral superior olive nucleus (LSO). These calyx-like synapses expressed the Ca2+- sensor Synaptotagmin-2 (Syt-2) and the Ca2+ binding protein Parvalbumin (PV), two markers of developing calyces of Held in vivo. Application of the BMP inhibitor LDN-193189 significantly inhibited the growth of calyx synapses, demonstrating the feasibility of long-term pharmacological manipulation using this organotypic culture method. These experiments provide a method for organotypic culturing of calyces of Held, and show that the formation of calyx-like synapses onto MNTB neurons can be preserved in-vitro. Furthermore, our study adds pharmacological evidence for a role of BMP-signaling in the formation of large calyx of Held synapses.

Nkx2.1 regulates the generation of telencephalic astrocytes during embryonic development.

The homeodomain transcription factor Nkx2.1 (NK2 homeobox 1) controls cell differentiation of telencephalic GABAergic interneurons and oligodendrocytes. Here we show that Nkx2.1 also regulates astrogliogenesis of the telencephalon from embryonic day (E) 14.5 to E16.5. Moreover we identify the different mechanisms by which Nkx2.1 controls the telencephalic astrogliogenesis. In Nkx2.1 knockout (Nkx2.1-/-) mice a drastic loss of astrocytes is observed that is not related to cell death. Further, in vivo analysis using BrdU incorporation reveals that Nkx2.1 affects the proliferation of the ventral neural stem cells that generate early astrocytes. Also, in vitro neurosphere assays showed reduced generation of astroglia upon loss of Nkx2.1, which could be due to decreased precursor proliferation and possibly defects in glial specification/differentiation. Chromatin immunoprecipitation analysis and in vitro co-transfection studies with an Nkx2.1-expressing plasmid indicate that Nkx2.1 binds to the promoter of glial fibrillary acidic protein (GFAP), primarily expressed in astrocytes, to regulate its expression. Hence, Nkx2.1 controls astroglial production spatiotemporally in embryos by regulating proliferation of the contributing Nkx2.1-positive precursors.

Synaptic Adhesion Molecules Regulate the Integration of New Granule Neurons in the Postnatal Mouse Hippocampus and their Impact on Spatial Memory.

Postnatal hippocampal neurogenesis induces network remodeling and may participate to mechanisms of learning. In turn, the maturation and survival of newborn neurons is regulated by their activity. Here, we tested the effect of a cell-autonomous overexpression of synaptic adhesion molecules on the maturation and survival of neurons born postnatally and on hippocampal-dependent memory performances. Families of adhesion molecules are known to induce pre- and post-synaptic assembly. Using viral targeting, we overexpressed three different synaptic adhesion molecules, SynCAM1, Neuroligin-1B and Neuroligin-2A in newborn neurons in the dentate gyrus of 7- to 9-week-old mice. We found that SynCAM1 increased the morphological maturation of dendritic spines and mossy fiber terminals while Neuroligin-1B increased spine density. In contrast, Neuroligin-2A increased both spine density and size as well as GABAergic innervation and resulted in a drastic increase of neuronal survival. Surprisingly, despite increased neurogenesis, mice overexpressing Neuroligin-2A in new neurons showed decreased memory performances in a Morris water maze task. These results indicate that the cell-autonomous overexpression of synaptic adhesion molecules can enhance different aspects of synapse formation on new neurons and increase their survival. Furthermore, they suggest that the mechanisms by which new neurons integrate in the postnatal hippocampus conditions their functional implication in learning and memory.

NG2 glia are required for vessel network formation during embryonic development.

The NG2(+) glia, also known as polydendrocytes or oligodendrocyte precursor cells, represent a new entity among glial cell populations in the central nervous system. However, the complete repertoire of their roles is not yet identified. The embryonic NG2(+) glia originate from the Nkx2.1(+) progenitors of the ventral telencephalon. Our analysis unravels that, beginning from E12.5 until E16.5, the NG2(+) glia populate the entire dorsal telencephalon. Interestingly, their appearance temporally coincides with the establishment of blood vessel network in the embryonic brain. NG2(+) glia are closely apposed to developing cerebral vessels by being either positioned at the sprouting tip cells or tethered along the vessel walls. Absence of NG2(+) glia drastically affects the vascular development leading to severe reduction of ramifications and connections by E18.5. By revealing a novel and fundamental role for NG2(+) glia, our study brings new perspectives to mechanisms underlying proper vessels network formation in embryonic brains.

Nkx2.1-derived astrocytes and neurons together with Slit2 are indispensable for anterior commissure formation.

Guidepost cells present at and surrounding the midline provide guidance cues that orient the growing axons through commissures. Here we show that the transcription factor Nkx2.1 known to control the specification of GABAergic interneurons also regulates the differentiation of astroglia and polydendrocytes within the mouse anterior commissure (AC). Nkx2.1-positive glia were found to originate from three germinal regions of the ventral telencephalon. Nkx2.1-derived glia were observed in and around the AC region by E14.5. Thereafter, a selective cell ablation strategy showed a synergistic role of Nkx2.1-derived cells, both GABAergic interneurons and astroglia, towards the proper formation of the AC. Finally, our results reveal that the Nkx2.1-regulated cells mediate AC axon guidance through the expression of the repellent cue, Slit2. These results bring forth interesting insights about the spatial and temporal origin of midline telencephalic glia, and highlight the importance of neurons and astroglia towards the formation of midline commissures.

Two specific populations of GABAergic neurons originating from the medial and the caudal ganglionic eminences aid in proper navigation of callosal axons.

The corpus callosum (CC) plays a crucial role in interhemispheric communication. It has been shown that CC formation relies on the guidepost cells located in the midline region that include glutamatergic and GABAergic neurons as well as glial cells. However, the origin of these guidepost GABAergic neurons and their precise function in callosal axon pathfinding remain to be investigated. Here, we show that two distinct GABAergic neuronal subpopulations converge toward the midline prior to the arrival of callosal axons. Using in vivo and ex vivo fate mapping we show that CC GABAergic neurons originate in the caudal and medial ganglionic eminences (CGE and MGE) but not in the lateral ganglionic eminence (LGE). Time lapse imaging on organotypic slices and in vivo analyses further revealed that CC GABAergic neurons contribute to the normal navigation of callosal axons. The use of Nkx2.1 knockout (KO) mice confirmed a role of these neurons in the maintenance of proper behavior of callosal axons while growing through the CC. Indeed, using in vitro transplantation assays, we demonstrated that both MGE- and CGE-derived GABAergic neurons exert an attractive activity on callosal axons. Furthermore, by combining a sensitive RT-PCR technique with in situ hybridization, we demonstrate that CC neurons express multiple short and long range guidance cues. This study strongly suggests that MGE- and CGE-derived interneurons may guide CC axons by multiple guidance mechanisms and signaling pathways.

The ciliogenic transcription factor RFX3 regulates early midline distribution of guidepost neurons required for corpus callosum development.

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3-deficient mice show several hallmarks of ciliopathies including left-right asymmetry defects and hydrocephalus. Here we show that Rfx3-deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.

Loss of ATF2 function leads to cranial motoneuron degeneration during embryonic mouse development.

The AP-1 family transcription factor ATF2 is essential for development and tissue maintenance in mammals. In particular, ATF2 is highly expressed and activated in the brain and previous studies using mouse knockouts have confirmed its requirement in the cerebellum as well as in vestibular sense organs. Here we present the analysis of the requirement for ATF2 in CNS development in mouse embryos, specifically in the brainstem. We discovered that neuron-specific inactivation of ATF2 leads to significant loss of motoneurons of the hypoglossal, abducens and facial nuclei. While the generation of ATF2 mutant motoneurons appears normal during early development, they undergo caspase-dependent and independent cell death during later embryonic and foetal stages. The loss of these motoneurons correlates with increased levels of stress activated MAP kinases, JNK and p38, as well as aberrant accumulation of phosphorylated neurofilament proteins, NF-H and NF-M, known substrates for these kinases. This, together with other neuropathological phenotypes, including aberrant vacuolisation and lipid accumulation, indicates that deficiency in ATF2 leads to neurodegeneration of subsets of somatic and visceral motoneurons of the brainstem. It also confirms that ATF2 has a critical role in limiting the activities of stress kinases JNK and p38 which are potent inducers of cell death in the CNS.

Transient neuronal populations are required to guide callosal axons: a role for semaphorin 3C.

The corpus callosum (CC) is the main pathway responsible for interhemispheric communication. CC agenesis is associated with numerous human pathologies, suggesting that a range of developmental defects can result in abnormalities in this structure. Midline glial cells are known to play a role in CC development, but we here show that two transient populations of midline neurons also make major contributions to the formation of this commissure. We report that these two neuronal populations enter the CC midline prior to the arrival of callosal pioneer axons. Using a combination of mutant analysis and in vitro assays, we demonstrate that CC neurons are necessary for normal callosal axon navigation. They exert an attractive influence on callosal axons, in part via Semaphorin 3C and its receptor Neuropilin-1. By revealing a novel and essential role for these neuronal populations in the pathfinding of a major cerebral commissure, our study brings new perspectives to pathophysiological mechanisms altering CC formation.

Expression of the NH(2)-terminal fragment of RasGAP in pancreatic beta-cells increases their resistance to stresses and protects mice from diabetes.

OBJECTIVE: Our laboratory has previously established in vitro that a caspase-generated RasGAP NH(2)-terminal moiety, called fragment N, potently protects cells, including insulinomas, from apoptotic stress. We aimed to determine whether fragment N can increase the resistance of pancreatic beta-cells in a physiological setting. RESEARCH DESIGN AND METHODS: A mouse line, called rat insulin promoter (RIP)-N, was generated that bears a transgene containing the rat insulin promoter followed by the cDNA-encoding fragment N. The histology, functionality, and resistance to stress of RIP-N islets were then assessed. RESULTS: Pancreatic beta-cells of RIP-N mice express fragment N, activate Akt, and block nuclear factor kappaB activity without affecting islet cell proliferation or the morphology and cellular composition of islets. Intraperitoneal glucose tolerance tests revealed that RIP-N mice control their glycemia similarly as wild-type mice throughout their lifespan. Moreover, islets isolated from RIP-N mice showed normal glucose-induced insulin secretory capacities. They, however, displayed increased resistance to apoptosis induced by a series of stresses including inflammatory cytokines, fatty acids, and hyperglycemia. RIP-N mice were also protected from multiple low-dose streptozotocin-induced diabetes, and this was associated with reduced in vivo beta-cell apoptosis. CONCLUSIONS: Fragment N efficiently increases the overall resistance of beta-cells to noxious stimuli without interfering with the physiological functions of the cells. Fragment N and the pathway it regulates represent, therefore, a potential target for the development of antidiabetes tools.

Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice.

Serotonin [5-hydroxytryptamine (5-HT)] neurotransmission in the central nervous system modulates depression and anxiety-related behaviors in humans and rodents, but the responsible downstream receptors remain poorly understood. We demonstrate that global disruption of 5-HT2A receptor (5HT2AR) signaling in mice reduces inhibition in conflict anxiety paradigms without affecting fear-conditioned and depression-related behaviors. Selective restoration of 5HT2AR signaling to the cortex normalized conflict anxiety behaviors. These findings indicate a specific role for cortical 5HT2AR function in the modulation of conflict anxiety, consistent with models of cortical, "top-down" influences on risk assessment.

Glutathione deficit during development induces anomalies in the rat anterior cingulate GABAergic neurons: Relevance to schizophrenia.

A series of studies in schizophrenic patients report a decrease of glutathione (GSH) in prefrontal cortex (PFC) and cerebrospinal fluid, a decrease in mRNA levels for two GSH synthesizing enzymes and a deficit in parvalbumin (PV) expression in a subclass of GABA neurons in PFC. GSH is an important redox regulator, and its deficit could be responsible for cortical anomalies, particularly in regions rich in dopamine innervation. We tested in an animal model if redox imbalance (GSH deficit and excess extracellular dopamine) during postnatal development would affect PV-expressing neurons. Three populations of interneurons immunolabeled for calcium-binding proteins were analyzed quantitatively in 16-day-old rat brain sections. Treated rats showed specific reduction in parvalbumin immunoreactivity in the anterior cingulate cortex, but not for calbindin and calretinin. These results provide experimental evidence for the critical role of redox regulation in cortical development and validate this animal model used in schizophrenia research.

Selective neurofilament (SMI-32, FNP-7 and N200) expression in subpopulations of layer V pyramidal neurons in vivo and in vitro.

There are two main types of layer V pyramidal neurons in rat cortex. Type I neurons have tufted apical dendrites extending into layer I, produce bursts of action potentials and project to subcortical targets (spinal cord, superior colliculus and pontine nuclei). Type II neurons have apical dendrites, which arborize in layers II-IV, do not produce bursts of action potentials and project to ipsilateral and contralateral cortex. The specific expression of different genes and proteins in these two distinct layer V neurons is unknown. To distinguish between distinct subpopulations, fluorescent microspheres were injected into subcortical targets (labeling type I neurons) or primary somatosensory cortex (labeling type II neurons) of adult rats. After transport, cortical sections were processed for immunohistochemistry using various antibodies. This study demonstrated that antigens recognized by SMI-32, N200 and FNP-7 antibodies were only expressed in subcortical (type I)--but not in contralateral (type II)--projecting neurons. NR1, NR2a/b, PLCbeta1, BDNF, NGF and TrkB antigens were highly expressed in all neuronal subpopulations examined. Organotypic culture experiments demonstrated that the development of neurofilament expression and laminar specificity does not depend on the presence of the subcortical targets. This study suggests specific markers for the subcortical projecting layer V neuron subpopulations.

Ligation of the inferior mesenteric artery in the surgery of rectal cancer: anatomical considerations.

BACKGROUND: No agreement has been found in the literature concerning the safest point of ligation of the inferior mesenteric artery (ima) in order to avoid nerve damage during the surgery of rectal cancer. STUDY DESIGN: The distance between the origin of the ima and the left paraortic trunk was measured, as was the distance between the left paraortic trunk and the origin of the left colic artery (lca). The measurements were carried out on 20 cadavers and during 22 operations for rectal cancer. RESULTS: The left paraortic trunk always runs posterior to the ima: its distance from the origin of the ima is on average 1.2 cm; the distance of the left paraortic trunk from the origin of the lca is on average 0.4 cm. The point at which the ima and the left paraortic trunk cross varies greatly, but it is never near the origin of the ima. CONCLUSIONS: From an anatomical point of view the safest point of ligation of the ima is at its origin. At this point, the left paraortic trunk never runs; so there isn't any risk to damage the nerve involving it during the ligation of the artery.

N-methyl-d-aspartate-triggered neuronal death in organotypic hippocampal cultures is endocytic, autophagic and mediated by the c-Jun N-terminal kinase pathway.

Acute excitotoxic neuronal death was studied in rat organotypic hippocampal slices exposed to 100 micro mN-methyl-d-aspartate. Fulgurant death of pyramidal neurons occurred in the CA1 and CA3 regions and was already detectable within 2 h of the N-methyl-d-aspartate administration. Morphologically, the neuronal death was neither apoptotic nor necrotic but had the hallmarks of autophagic neuronal death, as shown by acid phosphatase histochemistry in both CA1 and CA3 and by electron microscopy in CA1. The dying neurons also manifested strong endocytosis of horseradish peroxidase or microperoxidase, occurring probably by a fluid phase mechanism, and followed, surprisingly, by nuclear entry. In addition to these autophagic and endocytic characteristics, there were indications that the c-Jun N-terminal kinase pathway was activated. Its target c-Jun was selectively phosphorylated in CA1, CA3 and the dentate gyrus and c-Fos, the transcription of which is under the positive control of c-Jun N-terminal kinase target Elk1, was selectively up-regulated in CA1 and CA3. All these effects, the neuronal death itself and the associated autophagy and endocytosis, were totally prevented by a cell-permeable inhibitor of the interaction between c-Jun N-terminal kinase and certain of its targets. These results show that pyramidal neurons undergoing excitotoxic death in this situation are autophagic and endocytic and that both the cell death and the associated autophagy and endocytosis are under the control of the c-Jun N-terminal kinase pathway.

Early fetal expression of GABA(B1) and GABA(B2) receptor mRNAs on the development of the rat central nervous system.

GABA(B) receptors are G-protein-coupled receptors that mediate slow onset and prolonged effects of GABA in the central nervous system (CNS). While they appear to influence developmental events, depending on where they are found at a synapse, little, if anything, is known as to the expression of GABA(B1) and GABA(B2) receptor mRNAs during the early developmental stages. We used in situ hybridization and RNase protection assays (RPA) to investigate the early fetal expression of GABA(B1) and GABA(B2) receptor mRNAs on the development of the rat CNS. Our in situ studies defined a pattern of early and strong GABA(B1) receptor mRNA expression in the spinal cord, medullar and cerebral cortex neuroepithelium of discrete brain regions on gestational day (GD) 11.5. On GD 12.5, GABA(B1) receptor mRNAs were found in the hippocampal formation, cerebral cortex, intermediate and posterior neuroepithelium, and the pontine neuroepithelium of whole brain. RPA results showed GABA(B1) receptor mRNA was intensely expressed on GD 11.5 and GD 12.5, when it was first detected in the ganglia, thalamus, and cerebellum. However, GABA(B2) receptor mRNA was not detected on GD 10.5, 11.5, or 12.5. We suggest that GABA(B1) receptor might have a role in the early fetal brain and spinal cord during pre- and post-synaptogenesis, neuronal maturation, proliferation, and migration, and may be more important than the GABA(B2) receptor in the early development of the rat CNS.

The human raphe nuclei and the serotonergic system.

The raphe nuclei are distributed near the midline of the brainstem along its entire rostro-caudal extension. The serotonergic neurons are their main neuronal components, although a proportion of them lie in subdivisions of the lateral reticular formation. They develop from mesopontine and medullary primordia, and the resulting grouping into rostral and caudal clusters is maintained into adulthood, and is reflected in the connectivity. Thus, the mesencephalon and rostral pons, neurons within the rostral raphe complex (caudal linear, dorsal raphe, and median raphe nuclei) project primarily to the forebrain. By contrast, in the caudal pons and medulla oblongata, neurons within the caudal raphe complex (raphe magnus, raphe obscurus, raphe pallidus nuclei and parts of the adjacent lateral reticular formation) project to the brainstem nuclei and to the spinal cord. The median raphe and dorsal raphe nuclei provide parallel and overlapping projections to many forebrain structures with axon fibers exhibiting distinct structural and functional characteristics. The caudal group of the serotonergic system projects to the brainstem, and, by three parallel projections, to the dorsal, intermediate and ventral columns in the spinal cord. The serotonergic axons arborize over large areas comprising functionally diverse targets. Some projections form classical chemical synapses while many do not, thus contributing to the so-called paracrine or volume transmission. The serotonergic projections participate in the regulation of different functional (motor, somatosensory, limbic) systems; and have been associated with a wide range of neuropsychiatric and neurological disorders. Finally, recent experimental data support the role of serotonin in modulating brain development, such that a dysfunction in serotonergic transmission during early life could lead to long lasting structural and functional alterations.