A comparative study of vascular injection fluids in fresh-frozen and embalmed human cadaver forearms.

Over the years, various vascular injection products have been developed to facilitate anatomical dissections. This study aimed to compare the most commonly used vascular injection products in fresh-frozen and formalin-embalmed cadaver specimens. An overview of the properties, advantages and limitations of each substance was given, and a comparison of vascular infusion procedures in both preservation methods was made. A literature search was performed in order to identify the most commonly used vascular injection products. Acrylic paint, latex, gelatin, silicone, Araldite F and Batson's No. 17 were selected for the study. One fresh-frozen and one embalmed cadaver forearm were infused with each injection product according to a uniform protocol. The curing time, skin- and subcutaneous tissue penetration, degree of filling of the arterial tree, extravasations, consistency of the injected vessels during dissection, and the costs of each injection fluid were noted. There was a large variation between the injection fluids in processing- and curing time, colour intensity, flexibility, fragility, elasticity, strength, toxicity and costs. All fluids were suitable for infusion. The penetration of injection fluid into the skin and subcutaneous tissue was significantly better in fresh-frozen specimens (P = 0.002 and P = 0.009, respectively), with significantly smaller branches casted (P = 0.004). Vascular infusion of fresh-frozen cadaver specimens results in a significantly better filled coloured arterial tree, enabling more detail to be achieved and smaller branches casted. The biomechanical properties of fresh-frozen soft tissues are less affected compared with formalin fixation. All the injection fluids studied are suitable for vascular infusion, but their different properties ensure that certain products and procedures are more suitable for specific study purposes.

Abnormal fat distribution in PMM2-CDG.

We hypothesize that abnormal fat distribution, a common feature of PMM2-CDG, is associated with abnormal perinatal hormone regulation. We assessed 32 cases with PMM2-CDG, for the comorbidity of hypoglycemia/hyperinsulinism and fat pads. Ninety percent of patients with hypoketotic hypoglycemia and/or hyperinsulinism had abnormal fat distribution, while normoglycemic patients showed this feature in 50% of the cases. This statistically significant difference suggests an etiological role of the insulin receptor in developing abnormal fat distribution in PMM2-CDG.

Glycosylation defects underlying fetal alcohol spectrum disorder: a novel pathogenetic model. "When the wine goes in, strange things come out" - S.T. Coleridge, The Piccolomini.

Fetal alcohol spectrum disorder (FASD) is an umbrella term used to describe the craniofacial dysmorphic features, malformations, and disturbances in growth, neurodevelopment and behavior occurring in individuals prenatally exposed to alcohol. Fetal alcohol syndrome (FAS) represents the severe end of this spectrum. Many pathophysiological mechanisms have hitherto been proposed to account for the disrupted growth and morphogenesis seen in FAS. These include impaired cholesterol-modification of the Sonic hedgehog morphogen, retinoic acid deficiency, lipoperoxidative damage due to alcohol-induced reactive oxygen species combined with reduced antioxidant defences, and malfunctioning cell adhesion molecules. In this report, we propose a completely novel concept regarding the pathogenesis of FAS. Based on our observation that transferrin isoelectric focusing (TIEF) - the most widely used screening tool for congenital disorders of glycosylation (CDG) - was transiently abnormal in a newborn with FAS and a confirmed maternal history of gestational alcohol abuse, we came to believe that FAS exemplifies a congenital disorder of glycosylation secondary to alcohol-inflicted disruption of (N-linked) protein glycosylation. Various pieces of evidence were found in the literature to substantiate this hypothesis. This observation implies, among others, that one might need to consider the possibility of maternal alcohol consumption in newborns with transient glycosylation abnormalities. We also present an integrated pathophysiological model of FAS, which incorporates all existing theories mentioned above as well as our novel concept. This model highlights the pivotal role of disrupted isoprenoid metabolism in the origination of FAS.

Effects of early-life stress on peripheral and central mitochondria in male mice across ages.

Exposure to early-life stress (ES) increases the vulnerability to develop metabolic diseases as well as cognitive dysfunction, but the specific biological underpinning of the ES-induced programming is unknown. Metabolic and cognitive disorders are often comorbid, suggesting possible converging underlying pathways. Mitochondrial dysfunction is implicated in both metabolic diseases and cognitive dysfunction and chronic stress impairs mitochondrial functioning. However, if and how mitochondria are impacted by ES and whether they are implicated in the ES-induced programming remains to be determined. ES was applied by providing mice with limited nesting and bedding material from postnatal day (P)2-P9, and metabolic parameters, cognitive functions and multiple aspects of mitochondria biology (i.e. mitochondrial electron transport chain (ETC) complex activity, mitochondrial DNA copy number, expression of genes relevant for mitochondrial function, and the antioxidant capacity) were studied in muscle, hypothalamus and hippocampus at P9 and late adulthood (10-12 months of age). We show that ES altered bodyweight (gain), adiposity and glucose levels at P9, but not in late adulthood. At this age, however, ES exposure led to cognitive impairments. ES affected peripheral and central mitochondria in an age-dependent manner. At P9, both muscle and hypothalamic ETC activity were affected by ES, while in hippocampus, ES altered the expression of genes involved in fission and antioxidant defence. In adulthood, alterations in ETC complex activity were observed in the hypothalamus specifically, whereas in muscle and hippocampus ES affected the expression of genes involved in mitophagy and fission, respectively. Our study demonstrates that ES affects peripheral and central mitochondria biology throughout life, thereby uncovering a converging mechanism that might contribute to the ES-induced vulnerability for both metabolic diseases and cognitive dysfunction, which could serve as a novel target for intervention.

Diurnal expression of period 2 and urocortin 1 in neurones of the non-preganglionic Edinger-Westphal nucleus in the rat.

Period 2 (Per2) is an important clock gene involved in the regulation of the major circadian clock in the mammalian central nervous system, the suprachiasmatic nucleus. In addition, Per2 is expressed in many other stress-sensitive brain structures. We have previously showed that the non-preganglionic Edinger-Westphal nucleus (npEW) is the main site of the corticotropin-releasing factor peptide family member urocortin 1 (Ucn1) and that this peptide undergoes conspicuous expression changes in response to various stressors. Here, we hypothesized that in the rat npEW both Per2 and Ucn1 would be produced in a diurnal, rhythmical fashion. This hypothesis was tested by following this expected rhythm on two days in rats killed at four time points each day (Zeitgeber times 0, 6, 12, and 18). We showed the co-existence of Per2 and Ucn1 in the npEW with double-label immunofluorescence and demonstrated with quantitative RT-PCR and semi-quantitative immunocytochemistry diurnal rhythms in Per2 mRNA expression and Per2 protein content, each on a single different day, with a minimum at lights-off and a maximum at lights-on. We furthermore revealed a diurnal rhythm in the number of Ucn1-immunopositive neurones and in their Ucn1 peptide content, with a minimum at night and at the beginning of the light period and a peak at lights-off, while the Ucn1 mRNA content paralleled the Per2 mRNA rhythm. The rhythms were accompanied by a diurnal rhythm in plasma corticosterone concentration. Our results are in line with the hypothesis that both Per2 and Ucn1 in the rat npEW are produced in a diurnal fashion, a phenomenon that may be relevant for the regulation of the diurnal rhythm in the stress response.

Gender-related urocortin 1 and brain-derived neurotrophic factor expression in the adult human midbrain of suicide victims with major depression.

In postmortem brains of patients with major depression, the expression of corticotrophin-releasing factor (CRF) is enhanced and that of brain-derived neurotrophic factor (BDNF) decreased. In mice over-expressing neuronal CRF (an animal model for depression) the expression of urocortin 1 (Ucn1) in the non-preganglionic Edinger-Westphal nucleus (npEW) is strongly down-regulated. Therefore, we hypothesized that an altered activity of Ucn1 neurons in the npEW would contribute to the pathogenesis of major depression. To test this hypothesis we measured Ucn1 mRNA and BDNF mRNA levels in the npEW of seven male and four female, drug-free suicide victims with major depression, and compared the data with those obtained from 10 male and seven female individuals without neurological and psychiatric disorders (controls). We show that compared with controls, the Ucn1-mRNA level in npEW neurons is about 9.12 times higher in male but unchanged in female suicide victims. Furthermore, BDNF mRNA expression in microdissections of npEW was 3.36 times lower in male suicide victims, but 5.27 times higher in female victims, compared with controls. Our data also show that male suicide victims had almost 11.47 times more Ucn1 and 4.26 times less BDNF mRNA in the npEW than female suicide victims. We discuss the significance of these data for npEW Ucn1 and BDNF, and propose that altered expressions of Ucn1 and BDNF in the npEW contribute to the pathogenesis of major depression and/or suicidality in a gender-specific manner.

Chronic psychosocial stress affects corticotropin-releasing factor in the paraventricular nucleus and central extended amygdala as well as urocortin 1 in the non-preganglionic Edinger-Westphal nucleus of the tree shrew.

Stressful stimuli evoke neuronal and neuroendocrine responses helping an organism to adapt to changed environmental conditions. Chronic stressors may induce maladaptive responses leading to psychiatric diseases, such as anxiety and major depression. A suitable animal model to unravel mechanisms involved in the control of adaptation to chronic stress is the psychological subordination stress in the male tree shrew. Subordinate male tree shrews exhibit chronic hypothalamo-pituitary-adrenal (HPA) activation as reflected in continuously elevated cortisol secretion, and structural changes in the hippocampal formation. Corticotropin-releasing factor (CRF) is the major peptide released upon activation of the HPA axis in response to stress. Recent evidence suggests that besides CRF, urocortin 1 (Ucn1) also plays a role in stress adaptation. We have tested the significance of CRF and Ucn1 in adaptation to chronic psychosocial stress in male tree shrews exposed for 35 days to daily psychosocial conflict, by performing semi-quantitative immunocytochemistry for CRF in the parvocellular hypothalamic paraventricular nucleus (pPVN), extended amygdala, viz. central extended amygdala (CeA) and dorsolateral nucleus of the bed nucleus of the stria terminalis (BNSTdl) as well as that for Ucn1 in the non-preganglionic Edinger-Westphal nucleus (npEW). Compared to unstressed animals, psychosocial stress resulted in an immediate and sustained activation of the HPA axis and sympathetic tone as well as reduced testosterone concentration and decreased body and testis weights vs. non-stressed tree shrews. In the pPVN, the number of CRF-immunoreactive neurons and the specific signal density of CRF-immunoreactive fiber terminals in the CeA were strongly reduced (-300 and -40%, respectively; P<0.05), whereas no significant difference in CRF fiber density was found in BNSTdl. The npEW revealed 4 times less Ucn1-immunoreactive neurons (P<0.05). These clear effects on both Ucn1- and CRF-neuropeptide contents may reflect a crucial mechanism enabling the animal to adapt successfully to the stressors, and point to the significance of the pPVN, CeA and npEW in stress-induced brain diseases.

Modulation of glucocorticoids by the serotonin transporter polymorphism: A narrative review.

The biological background and consequences of serotonin transporter polymorphism-glucocorticoid relationship in individual differences in stress reactivity has been a major interest in neuropsychiatry research. Individual differences in glucocorticoid release have long been implicated in vulnerability to stress-related psychopathologies, like depression and anxiety in various species. Yet, it is largely elusive to what extent results from non-human primates and rodents translate to human findings. Based on our structured, comprehensive and non-hypothesis driven overview of this topic, we conclude that although gene-environment interaction studies have highlighted the importance of serotonin transporter polymorphism in modulating glucocorticoid release, there is compelling evidence that age, gender and ethnicity are significant factors too contributing to the equation. We conclude too that the way early life events modulate an individual's stress reactivity as a function of serotonin transporter polymorphism is comparable between species. These data provide a rationale for the design of new, prospective translational studies into sex-specific gene-environment interactions across the lifespan with the goal of improving preventative efforts and optimizing (personalized) treatment in stress-related psychopathologies.

Epigenetic programming of the neuroendocrine stress response by adult life stress.

The hypothalamic-pituitary-adrenal (HPA) axis is critically involved in the neuroendocrine regulation of stress adaptation, and the restoration of homeostasis following stress exposure. Dysregulation of this axis is associated with stress-related pathologies like major depressive disorder, post-traumatic stress disorder, panic disorder and chronic anxiety. It has long been understood that stress during early life can have a significant lasting influence on the development of the neuroendocrine system and its neural regulators, partially by modifying epigenetic regulation of gene expression, with implications for health and well-being in later life. Evidence is accumulating that epigenetic plasticity also extends to adulthood, proposing it as a mechanism by which psychological trauma later in life can long-lastingly affect HPA axis function, brain plasticity, neuronal function and behavioural adaptation to neuropsychological stress. Further corroborating this claim is the phenomenon that these epigenetic changes correlate with the behavioural consequences of trauma exposure. Thereby, epigenetic modifications provide a putative molecular mechanism by which the behavioural phenotype and transcriptional/translational potential of genes involved in HPA axis regulation can change drastically in response to environmental challenges, and appear an important target for treatment of stress-related disorders. However, improved insight is required to increase their therapeutic (drug) potential. Here, we provide an overview of the growing body of literature describing the epigenetic modulation of the (primarily neuroendocrine) stress response as a consequence of adult life stress and interpret the implications for, and the challenges involved in applying this knowledge to, the identification and treatment of stress-related psychiatric disorders.

Radiological imaging of teratological fetuses: what can we learn?

OBJECTIVES: To determine the advantages of radiological imaging of a collection of full-term teratological fetuses in order to increase their scientific and educational value. BACKGROUND : Anatomical museums around the world exhibit full-term teratological fetuses. Unfortunately, these museums are regularly considered as "morbid cabinets". Detailed dysmorphological information concerning the exhibited specimens is often lacking. Moreover, fetuses with severe and complex congenital anomalies are frequently diagnosed incompletely, incorrectly or not at all. METHODS: In order to verify diagnoses and to enrich their educational and scientific value, we imaged 41 out of the 72 teratological specimens present in the collection of our Anatomy and Pathology Museum in Nijmegen (The Netherlands) by means of magnetic resonance imaging (MRI) and computed tomography (CT). Additionally, contemporary dysmorphological insights and 3D models are implemented in the teratology education of medical students and residents. CONCLUSIONS: Full-term teratological fetuses have become increasingly rare and deserve a prominent place in every anatomical museum; they are suitable for contemporary teratological research and education. Modern radiological techniques markedly enhance their scientific and didactic value. TEACHING POINTS: • To explore the scientific and educational potential of institutionalised teratological collections • To understand the additional value of radiological imaging in diagnosing teratological specimens • To learn about the specific settings of MRI parameters when scanning fixed specimens • To recognise specific internal dysmorphology in several congenital anomalies.

Distribution of urocortin-like immunoreactivity in the central nervous system of the rat.

Urocortin was recently cloned from the rat midbrain. Urocortin is a member of the corticotropin releasing factor (CRF) peptide family and shows 45% sequence identity to CRF and 63% sequence identity to urotensin. It binds with a high affinity to CRF1 and CRF2 receptors, resulting in the stimulation of their adenylate cyclase activity. We used a polyclonal antibody against rat urocortin to define the distribution of urocortin-like immunoreactivity in the rat central nervous system. Several immunostained cell bodies were found in the supraoptic, paraventricular, and ventromedial hypothalamic nuclei. A large number of neurons with urocortin-like immunoreactivity were seen in the dorsolateral tegmental nucleus, in the linear and dorsal raphe nuclei, and in the substantia nigra. The most abundant immunoreactive (ir) perikarya were found in the Edinger-Westphal nucleus. Some neurons showed immunoreactivity in the interstitial nucleus of Cajal, the nucleus of Darkeschewitsch, and the periaqueductal gray. A dense immunoreactive fiber network was found in the lateral septal area. Some faintly stained axon terminals were observed among urocortin-ir perikarya in the supraoptic and paraventricular nuclei, in the central and periaqueductal gray, and in the Edinger-Westphal nucleus. No fibers with urocortin-ir were seen in the median eminence or the posterior pituitary. The distribution of urocortin-ir overlapped with the expression of the mRNA for the CRF2 receptor in several brain areas. These data support the hypothesis that this peptide is the endogenous ligand for the CRF2 receptor. Urocortin has been implicated in various endocrine responses, such as blood pressure regulation, as well as in higher cognitive functions.

Distribution of neuromedin U-like immunoreactivity in the central nervous system of Rana esculenta.

The distribution of perikarya and nerve fibers containing neuromedin U-like immunoreactivity in the brain of Rana esculenta was determined with an antiserum directed toward the carboxyl terminus of the peptide. In the telencephalon, immunoreactive perikarya were found in the olfactory bulb, the medial septum, and the diagonal band. In the diencephalon, labeled perikarya were detected in the anterior and posterior preoptic areas, the dorsal nucleus of the hypothalamus, the caudal part of the infundibulum, and the posterior tuberculum. In the mesencephalon, immunoreactive cell bodies were found only in the laminar nucleus of the torus semicircularis and the anterodorsal tegmental nucleus. In the rhombencephalon, labeled perikarya were detected in the secondary visceral nucleus, the cerebellar nucleus, the central gray, and the nucleus of the solitary tract. Immunoreactive nerve fibers were observed in all areas of the brain that contained labeled perikarya. The densest accumulations were found in the nucleus accumbens; the dorsal part of the lateral septum; the periventricular region of the ventral thalamus; the lateral part of the infundibulum; the anterodorsal, anteroventral, posterodorsal, and posteroventral tegmental nuclei; and the periaqueductal region of the tegmentum. The distribution of neuromedin U-like immunoreactivity in the frog brain was substantially different from the distribution described for the rodent brain.

Distribution of proneuropeptide Y-derived peptides in the brain of Rana esculenta and Xenopus laevis.

The distribution of proneuropeptide Y-containing perikarya and nerve fibers in the brain of Rana esculenta and Xenopus laevis was determined with antisera directed toward neuropeptide Y and the carboxyl terminal flanking peptide. The distribution of proneuropeptide Y-like immunoreactivity was similar in both anurans. In the telencephalon, immunoreactive perikarya were found in the olfactory bulb, all subdivisions of the pallium, the septum, pars lateralis of the amygdala, the nucleus accumbens, and the anterior preoptic area. In the diencephalon, labelled perikarya were detected in the ventromedial, ventrolateral and central thalamic nuclei, the magnocellular preoptic nucleus, the suprachiasmatic nucleus, the posterior tuberculum, and the infundibulum. Amacrine-like cells were stained in the retina. In the pretectal area, posterior thalamic neurons showed intense, Golgi-like immunostaining. In the mesencephalon, immunoreactive cells were found in the reticular nucleus, the anteroventral tegmental nucleus, the optic tectum, the interpeduncular nucleus, and the torus semicircularis. In the rhombencephalon, labelled perikarya were detected in the secondary visceral nucleus, the central gray, the nucleus of the solitary tract, the dorsal column nuclei, and the spinal nucleus of the trigeminal nerve. Immunoreactive nerve fibers were observed in all areas of the brain that contained labelled perikarya. The densest accumulations were found in the accessory olfactory bulb, pars lateralis of the amygdala, the ventral habenula, the posterior pituitary, the optic tectum, the interpeduncular nucleus, and the saccular nucleus. The distribution of proneuropeptide Y-like immunoreactivity in the anuran brain showed many similarities to the distribution described for the amniote brain.

Neurons colocalizing urocortin and cocaine and amphetamine-regulated transcript immunoreactivities are induced by acute lipopolysaccharide stress in the Edinger-Westphal nucleus in the rat.

Besides corticotropin releasing factor, central stress regulatory pathways utilize various neurotransmitters/neuropeptides, such as urocortin and cocaine and amphetamine-regulated transcript, which play an important role in modifying the efferent components of endocrine, immune and behavioral responses to stress. Urocortin's distribution in the rat's brain has been demonstrated, with the most abundant urocortin-ir perikarya present in Edinger-Westphal nucleus. Cocaine and amphetamine-regulated transcript is widely expressed in the rat brain, with a dominant seat of cellular expression also in the Edinger-Westphal nucleus. Since immediate early gene expressions were seen in several midbrain regions, such as in the Edinger-Westphal nucleus, following various acute stresses, the Edinger-Westphal nucleus has been postulated to exert a regulatory/modulatory control over stress responses. Based on these data we decided to investigate the possible colocalization of urocortin and cocaine and amphetamine-regulated transcript-ir in the Edinger-Westphal nucleus using semithin double-label immunofluorescence technique. Furthermore, we also studied whether urocortinergic neurons colocalizing with cocaine and amphetamine-regulated transcript are recruited by lipopolysaccharide stress. Our experiments revealed that urocortin and cocaine and amphetamine-regulated transcript immunoreactivities colocalize in the Edinger-Westphal nucleus. In addition, our studies using the inducible immediate early gene c-fos as a marker of activated neurons demonstrated a significant stress-induced activation in perikarya colocalizing urocortin- and cocaine and amphetamine-regulated transcript-ir in the Edinger-Westphal nucleus. In view of these data it can be postulated that neurons colocalizing cocaine and amphetamine-regulated transcript and urocortin immunoreactivities respond to acute stress, and may play a role in modulating various physiological functions, such as feeding behaviors.

Cocaine- and amphetamine-regulated transcript peptide (CART) is a selective marker of rat granule cells and of human mossy cells in the hippocampal dentate gyrus.

Cocaine- and amphetamine-regulated transcript (CART) peptide immunocytochemistry was used to reveal cellular localization in the dentate gyrus and in Ammon's horn of the rat and human hippocampal formations. In the rat dentate gyrus, only granule cells were labeled, whereas in humans, only mossy cells of the hilar region expressed CART peptide immunoreactivity. In the rat, CART-positive granule cells were located at the molecular layer border of the granule cell layer and had no features that would distinguish them from other granule cells. The mossy fiber bundle was labeled in the hilus as well as along the entire CA3 area of Ammon's horn. In the human, CART-immunoreactive mossy cells displayed the characteristic thorny excrescences both on their somata and their main dendrites. Axon collaterals of mossy cells could be seen in the hilus and the main axons formed a dense band in the inner molecular layer of the dentate gyrus, suggesting that mossy cells are the principal source of the associational pathway. Granule cells of the dentate gyrus and pyramidal neurons of the human hippocampal formation were devoid of CART peptide immunoreactivity. A few labeled non-pyramidal cells and a large group of strongly immunostained axons of unknown origin were present in all layers of CA1-3. Granule cells are the main excitatory cell population of the dentate gyrus while mossy cells are in a key position in controlling activity of granule cells. The specific location of CART peptide in the dentate granule cells of rodents and in the mossy cells of the human hippocampus may indicate involvement of neuronal circuitry of the dentate gyrus in the memory-related effects of cocaine and amphetamine. Independently of its functional role, CART peptide can be used as a specific marker of human mossy cells and of the dentate associational pathway. The sensitivity of CART peptide to postmortem autolysis may restrict the use of this marker in surgically removed hippocampi or in human brains removed and fixed shortly after death.

Urocortin expression in the Edinger-Westphal nucleus is down-regulated in transgenic mice over-expressing neuronal corticotropin-releasing factor.

In recent years a large body of evidence has emerged linking chronic stress with increased vulnerability for depression and anxiety disorders. As corticotropin-releasing factor (CRF) is hypersecreted under these psychological conditions, we used our CRF-overexpressing (CRF-OE) mouse line to study underlying brain mechanisms possibly causing these disorders. Urocortin (Ucn), a recently discovered member of the CRF peptide family may play a role in the pathophysiology of stress-induced disorders. Stressors recruit Ucn-immunoreactive neurons in the Edinger-Westphal nucleus (E-WN), which is the major site of Ucn expression. Furthermore, E-WN Ucn mRNA levels are upregulated in CRF-deficient mice. Based on these findings, we hypothesized the down-regulation of E-WN Ucn in CRF-OE mice and consequently, altered responsiveness to stressful stimuli. Our results support this hypothesis as we found weaker immunohistochemical labeling with anti-Ucn and a six times weaker Ucn mRNA signal in E-WN in CRF-OE mice. Moreover, E-WN Ucn-expressing neurons mounted a response to acute challenge in CRF-OE mice too. From these results it is concluded that the CRF and E-WN Ucn neuronal systems work in concert in response to acute challenges, but are inversely regulated in their activities during chronic hyperactivity of the hypothalamo-pituitary-adrenal axis.

Colocalization of GABA, enkephalin and neuropeptide Y in the tectum of the green frog Rana esculenta.

The colocalization of GABA, enkephalin and neuropeptide Y immunoreactivity in neurons in the pretectal area and in the mesencephalic tectum of the green frog (Rana esculenta) was studied. Several Met-enkephalin immunoreactive perikarya were found in layer 6 of the tectum and every third of these neurons showed GABA-ir as well. Colocalization of GABA and NPY could also be shown in half of the neuropeptide Y immunopositive cells in the 6th layer of the tectum, but only a few cells were double stained in layers 9 and 4. In the pretectal area no colocalization of the investigated peptides and GABA was found.