RESUMEN
Different inbred mouse strains vary substantially in their behavior and metabolic phenotype under physiological and pathological conditions. The purpose of this study was to extend the knowledge of distinct coping strategies under challenging events in two differently adapting mouse strains: C57BL/6NTac (Bl6) and 129S6/SvEvTac (129Sv). Thus, we aimed to investigate possible similarities and differences in the body weight change, behavior, and several metabolic variables in Bl6 and 129Sv strains in response to high-fat diet (HFD) using the AbsoluteIDQ p180 kit. We found that 9 weeks of HFD induced a significant body weight gain in 129Sv, but not in Bl6 mice. Besides that, 129Sv mice displayed anxiety-like behavior in the open-field test. Metabolite profiling revealed that 129Sv mice had higher levels of circulating branched-chain amino acids, which were even more amplified by HFD. HFD also induced a decrease in glycine, spermidine, and t4-OH-proline levels in 129Sv mice. Although acylcarnitines (ACs) dominated in baseline conditions in 129Sv strain, this strain had a significantly stronger AC-reducing effect of HFD. Moreover, 129Sv mice had higher levels of lipids in baseline conditions, but HFD caused more pronounced alterations in lipid profile in Bl6 mice. Taken together, our results show that the Bl6 line is better adapted to abundant fat intake.
Asunto(s)
Dieta Alta en Grasa , Espermidina , Aminoácidos de Cadena Ramificada , Animales , Dieta Alta en Grasa/efectos adversos , Glicina , Lípidos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Prolina , Aumento de PesoRESUMEN
Ischemia reperfusion injury is common in transplantation. Previous studies have shown that cooling can protect against hypoxic injury. To date, the protective effects of hypothermia have been largely associated with metabolic suppression. Since kidney transplantation is one of the most common organ transplant surgeries, we used human-derived renal proximal tubular cells (HKC8 cell line) as a model of normal renal cells. We performed a temperature titration curve from 37 °C to 22 °C and evaluated cellular respiration and molecular mechanisms that can counteract the build-up of reducing equivalents in hypoxic conditions. We show that the protective effects of hypothermia are likely to stem both from metabolic suppression (inhibitory component) and augmentation of stress tolerance (activating component), with the highest overlap between activating and suppressing mechanisms emerging in the window of mild hypothermia (32 °C). Hypothermia decreased hypoxia-induced rise in the extracellular lactate:pyruvate ratio, increased ATP/ADP ratio and mitochondrial content, normalized lipid content, and improved the recovery of respiration after anoxia. Importantly, it was observed that in contrast to mild hypothermia, moderate and deep hypothermia interfere with HIF1 (hypoxia inducible factor 1)-dependent HRE (hypoxia response element) induction in hypoxia. This work also demonstrates that hypothermia alleviates reductive stress, a conceptually novel and largely overlooked phenomenon at the root of ischemia reperfusion injury.
Asunto(s)
Hipotermia Inducida , Hipotermia , Daño por Reperfusión , Frío , Humanos , HipoxiaRESUMEN
Depression is associated with abnormal lipid metabolism, and omega (n)-3 polyunsaturated fatty acids (PUFAs) can effectively treat depression. However, mechanism of lipid metabolism involved in the depressive attenuation remains poorly understood. Olfactory bulbectomy (OB)-induced changes in animal behavior and physiological functions are similar to those observed in depressed patients. Therefore, the present study used wild type (WT) and Fat-1 mice with or without OB to explore whether endogenous n-3 PUFA treatment of depression was through rectifying lipid metabolism, and to discover the possible lipid metabolic pathways. In WT mice, OB enhanced locomotor activity associated with up-regulation of lipid metabolites in the serum, such as phosphatidylcholines, L-a-glutamyl-L-Lysine and coproporphyrinogen III (Cop), which were involved in anti-inflammatory lipid metabolic pathways. OB also increased microglia activation marker CD11b and pro-inflammatory cytokines in the hippocampus. In one of the lipid pathways, increased Cop was significantly correlated with the hyper-activity of the OB mice. These OB-induced changes were markedly attenuated by endogenous n-3 PUFAs in Fat-1 mice. Additionally, increased expressions of anti-inflammatory lipid genes, such as fatty acid desaturase (Fads) and phospholipase A2 group VI (Pla2g6), were found in the hippocampus of Fat-1 mice compared with WT mice. Furthermore, Cop administration increased the production of pro-inflammatory cytokines and nitric oxide in a microglial cell line BV2. In conclusion, endogenous n-3 PUFAs in Fat-1 mice attenuated abnormal behavior in the depression model through restoration of lipid metabolism and suppression of inflammatory response.
Asunto(s)
Ácidos Grasos Omega-3 , Animales , Citocinas , Ácido Graso Desaturasas/genética , Ácidos Grasos Insaturados , Fosfolipasas A2 Grupo VI , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroglíaRESUMEN
Glucagon-like peptide 1 receptor agonists (GLP-1 RAs) are antidiabetic drugs with effects beyond antihyperglycemic action. The aim of the study was to examine whether a single dose of exenatide could be used as a stimulation test for the pituitary-adrenal axis. We carried out a single-group, open-label pilot clinical trial in an ambulatory setting. Ten healthy volunteers of both sexes with body weight>65 kg and age between 18-50 years were recruited. After fasting for 12 hours the subjects received 10 µg of exenatide solution subcutaneously. Blood samples were taken before the administration of exenatide and up to 150 minutes thereafter. The primary outcome was the maximal level of cortisol after the administration of exenatide. Single administration of exenatide 10 µg resulted in a modest increase in ACTH and cortisol levels, as compared to untreated values, and a decrease in blood glucose levels. Remarkably, a robust suppression of both renin and aldosterone levels occurred. We showed that acute administration of exenatide in a full therapeutic dose modestly stimulates the hypothalamic-pituitary-adrenal axis but inhibits the renin-aldosterone system. Further research is warranted to confirm this finding in the placebo-controlled study.
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Aldosterona/sangre , Exenatida/administración & dosificación , Receptor del Péptido 1 Similar al Glucagón/agonistas , Hidrocortisona/sangre , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Adolescente , Adulto , Aldosterona/química , Femenino , Estudios de Seguimiento , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
The melanocortin system is a major regulator of stress responses in the skin and is responsible for the induction of melanin synthesis through activation of melanogenesis enzymes. The expression of both melanocortin system genes and melanogenesis enzyme genes is altered in psoriasis, and the focus here was on twelve genes related to the signal transduction between them. Additionally, five endogenous opioid system genes that are involved in cutaneous inflammation were examined. Quantitative real-time-PCR was utilized to measure mRNA expression in punch biopsies from lesional and non-lesional skin of psoriasis patients and from the skin of healthy control subjects. Most of the genes related to melanogenesis were down-regulated in patients (CREB1, MITF, LEF1, USF1, MAPK14, ICAM1, PIK3CB, RPS6KB1, KIT, and ATRN). Conversely, an up-regulation occurred in the case of opioids (PENK, PDYN, and PNOC). The suppression of genes related to melanogenesis is in agreement with the reported reduction in pigmentation signaling in psoriatic skin and potentially results from the pro-inflammatory environment. The increase in endogenous opioids can be associated with their involvement in inflammatory dysregulation in psoriasis.
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Psoriasis/genética , Psoriasis/patología , Pigmentación de la Piel/genética , Adolescente , Adulto , Analgésicos Opioides/metabolismo , Biopsia , Estudios de Casos y Controles , Fosfatidilinositol 3-Quinasa Clase I/genética , Encefalinas/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Factor de Transcripción Asociado a Microftalmía/genética , Precursores de Proteínas/genética , Receptores Opioides/genética , Piel/patología , Adulto Joven , Receptor de NociceptinaRESUMEN
The members of the IgLON superfamily of cell adhesion molecules facilitate fundamental cellular communication during brain development, maintain functional brain circuitry, and are associated with several neuropsychiatric disorders such as depression, autism, schizophrenia, and intellectual disabilities. Usage of alternative promoter-specific 1a and 1b mRNA isoforms in Lsamp, Opcml, Ntm, and the single promoter of Negr1 in the mouse and human brain has been previously described. To determine the precise spatiotemporal expression dynamics of Lsamp, Opcml, Ntm isoforms, and Negr1, in the developing brain, we generated isoform-specific RNA probes and carried out in situ hybridization in the developing (embryonic, E10.5, E11.5, 13.5, 17; postnatal, P0) and adult mouse brains. We show that promoter-specific expression of IgLONs is established early during pallial development (at E10.5), where it remains throughout its differentiation through adulthood. In the diencephalon, midbrain, and hindbrain, strong expression patterns are initiated a few days later and begin fading after birth, being only faintly expressed during adulthood. Thus, the expression of specific IgLONs in the developing brain may provide the means for regionally specific functionality as well as for specific regional vulnerabilities. The current study will therefore improve the understanding of how IgLON genes are implicated in the development of neuropsychiatric disorders.
Asunto(s)
Encéfalo/embriología , Moléculas de Adhesión Celular/metabolismo , Regiones Promotoras Genéticas/genética , Animales , Encéfalo/metabolismo , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Hipocampo/embriología , Hipocampo/metabolismo , Inmunohistoquímica , Hibridación in Situ , Masculino , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Ratones , Ratones Endogámicos C57BL , Prosencéfalo/embriología , Prosencéfalo/metabolismo , Médula Espinal/embriología , Médula Espinal/metabolismoRESUMEN
Alterations in complex lipids may be involved in pathophysiology of schizophrenia spectrum disorders. Previously, we demonstrated importance of detecting lipid metabolism dysregulation by acylcarnitine (ACs) profile analysis in patients with first-episode psychosis (FEP). The aim of this study was to adopt lipidomics to identify serum glycerophospholipids (GPLs) and sphingomyelins (SMs) for describing FEP status before and after 7-month antipsychotic treatment. Using mass spectrometry and liquid chromatography technique, we profiled 105 individual lipids [14 lysophosphatidylcholines (LysoPCs), 76 phosphatidylcholines (PCs) and 15 SMs] in serum samples from 53 antipsychotic-naïve FEP patients, 44 of them were studied longitudinally and from 37 control subjects (CSs). Among the identified and quantified metabolites one LysoPC was elevated, and contrary the levels of 16 PCs as well as the level of one SM were significantly (p ≤ 0.0005) reduced in antipsychotic-naïve FEP patients compared to CSs. Comparison of serum lipids profiles of FEP patients before and after 7-month antipsychotic treatment revealed that 11 GPLs (2 LysoPCs, 9 PCs), and 2 SMs were found to be significantly changed (p ≤ 0.0005) in which GPLs were up-regulated, and SMs were down-regulated. However, no significant differences were noted when treated patient's serum lipid profiles were compared with CSs. Our findings suggest that complex lipid profile abnormalities are specifically associated with FEP and these discrepancies reflect two different disease-related pathways. Our findings provide insight into lipidomic information that may be used for monitoring FEP status and impact of the treatment in the early stage of the schizophrenia spectrum disorder.
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Antipsicóticos/farmacología , Glicerofosfolípidos/sangre , Lipidómica , Trastornos Psicóticos/sangre , Trastornos Psicóticos/tratamiento farmacológico , Esquizofrenia/sangre , Esquizofrenia/tratamiento farmacológico , Esfingomielinas/sangre , Adolescente , Adulto , Cromatografía Liquida , Femenino , Humanos , Estudios Longitudinales , Masculino , Espectrometría de Masas , Adulto JovenRESUMEN
Deficiency of the protein Wolfram syndrome 1 (WFS1) is associated with multiple neurological and psychiatric abnormalities similar to those observed in pathologies showing alterations in mitochondrial dynamics. The aim of this study was to examine the hypothesis that WFS1 deficiency affects neuronal function via mitochondrial abnormalities. We show that down-regulation of WFS1 in neurons leads to dramatic changes in mitochondrial dynamics (inhibited mitochondrial fusion, altered mitochondrial trafficking, and augmented mitophagy), delaying neuronal development. WFS1 deficiency induces endoplasmic reticulum (ER) stress, leading to inositol 1,4,5-trisphosphate receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn, alters mitochondrial dynamics. Importantly, ER stress, impaired Ca2+ homeostasis, altered mitochondrial dynamics, and delayed neuronal development are causatively related events because interventions at all these levels improved the downstream processes. Our data shed light on the mechanisms of neuronal abnormalities in Wolfram syndrome and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases.
Asunto(s)
Mitocondrias/metabolismo , Dinámicas Mitocondriales , Neurogénesis , Neuronas/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/citología , Encéfalo/metabolismo , Calcio/metabolismo , Células Cultivadas , Estrés del Retículo Endoplásmico/genética , Transferencia Resonante de Energía de Fluorescencia , Homeostasis , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Potencial de la Membrana Mitocondrial/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Microscopía Confocal , Mitocondrias/genética , Mitofagia/genética , Neuronas/citología , Células PC12 , Interferencia de ARN , Ratas , Ratas Wistar , Imagen de Lapso de Tiempo/métodos , Síndrome de Wolfram/genética , Síndrome de Wolfram/metabolismoRESUMEN
Acylcarnitines (ACs) have been shown to have a potential to activate pro-inflammatory signaling pathways and to foster the development of insulin resistance. The first task of the current study was to study the full list of ACs (from C2 to C18) in first episode psychosis (FEP) patients before and after antipsychotic treatment. The second task was to relate ACs to inflammatory and metabolic biomarkers established in the same patient cohort as in our previous studies. Serum levels of ACs were determined with the AbsoluteIDQ p180 kit (BIOCRATES Life Sciences AG, Innsbruck, Austria) using the flow injection analysis tandem mass spectrometry ([FIA]-MS/MS) as well as liquid chromatography ([LC]-MS/MS) technique. Identification and quantification of the metabolites was achieved using multiple reactions monitoring along with internal standards. The comparison of ACs in antipsychotic-naïve first-episode psychosis (FEP) patients (N = 38) and control subjects (CSs, N = 37) revealed significantly increased levels of long-chain ACs (LCACs) C14:1 (p = 0.0001), C16 (p = 0.00002), and C18:1 (p = 0.000001) in the patient group. These changes of LCACs were associated with augmented levels of CARN palmitoyltransferase 1 (CPT-1) (p = 0.006). By contrast, the level of short-chain AC (SCAC) C3 was significantly reduced (p = 0.00003) in FEP patients. Seven months of antipsychotic drug treatment ameliorated clinical symptoms in patients (N = 36) but increased significantly their body mass index (BMI, p = 0.001). These changes were accompanied by significantly reduced levels of C18:1 (p = 0.00003) and C18:2 (p = 0.0008) as well as increased level of C3 (p = 0.01). General linear model revealed the relation of LCACs (C16, C16:1, and C18:1) to the inflammatory markers (epidermal growth factor, IL-2, IL-4, IL-6), whereas SCAC C3 was linked to the metabolic markers (leptin, C-peptide) and BMI. FEP was associated with an imbalance of ACs in patients because the levels of several LCACs were significantly higher and the levels of several SCACs were significantly reduced compared with CSs. This imbalance was modified by 7 months of antipsychotic drug treatment, reversing the levels of both LCACs and SCACs to that established for CSs. This study supports the view that ACs have an impact on both inflammatory and metabolic alterations inherent for FEP.
Asunto(s)
Biomarcadores/sangre , Carnitina/análogos & derivados , Trastornos Psicóticos/sangre , Trastornos Psicóticos/tratamiento farmacológico , Adolescente , Adulto , Antipsicóticos/administración & dosificación , Índice de Masa Corporal , Carnitina/sangre , Carnitina/genética , Cromatografía Liquida , Femenino , Humanos , Resistencia a la Insulina/genética , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Masculino , Metabolómica , Persona de Mediana Edad , Trastornos Psicóticos/genética , Trastornos Psicóticos/patología , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
Nowadays, GLP-1 receptor agonists are widely used as effective and safe antidiabetic medications. In addition to glucose-dependent insulin secretion, their effects reach beyond glucose control. Previously, it has been shown that acute administration of GLP-1 receptor agonists increases circulating glucocorticoid and mineralocorticoid levels in both humans and rodents. So far, no studies have reported the effects of chronic administration of GLP-1 receptor agonists on the hypothalamic-pituitary-adrenal axis in humans. The aim of the current study was to examine the effects of acute and chronic treatment with the GLP-1 receptor agonist liraglutide on adrenal function in humans. Ten healthy volunteers were recruited into a single group open-label clinical trial. Each participant was tested for baseline levels, and after acute and chronic treatment with 0.6 mg liraglutide daily. A graded glucose infusion test was performed 3 times. We found that aldosterone tended to be suppressed (albeit not statistically different) after acute administration of liraglutide, and increased after chronic dosing; the difference was statistically significant when compared between acute and chronic dosing. Changes in aldosterone levels followed the changes in renin concentrations and the aldosterone-to-renin ratio remained stable. No statistically significant differences were observed in ACTH or cortisol levels. In conclusion, we have shown that a low dose of GLP-1 receptor agonist may interfere with renin and aldosterone release. Further studies in a larger patient sample and with higher doses of GLP-1 receptor agonists are warranted to corroborate this finding. The study protocol was registered at clinical.trials.gov (NCT02089256) and EU Clinical Trial Register (2014-000238-43).
Asunto(s)
Aldosterona/metabolismo , Hipoglucemiantes/farmacología , Liraglutida/farmacología , Sistema Renina-Angiotensina/efectos de los fármacos , Renina/metabolismo , Adolescente , Adulto , Femenino , Péptido 1 Similar al Glucagón/agonistas , Voluntarios Sanos , Humanos , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/metabolismo , Masculino , Persona de Mediana Edad , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/metabolismo , Potasio/sangre , Sodio/sangre , Adulto JovenRESUMEN
Regardless of the advent of high-throughput sequencing, microarrays remain central in current biomedical research. Conventional microarray analysis pipelines apply data reduction before the estimation of differential expression, which is likely to render the estimates susceptible to noise from signal summarization and reduce statistical power. We present a probe-level framework, which capitalizes on the high number of concurrent measurements to provide more robust differential expression estimates. The framework naturally extends to various experimental designs and target categories (e.g. transcripts, genes, genomic regions) as well as small sample sizes. Benchmarking in relation to popular microarray and RNA-sequencing data-analysis pipelines indicated high and stable performance on the Microarray Quality Control dataset and in a cell-culture model of hypoxia. Experimental-data-exhibiting long-range epigenetic silencing of gene expression was used to demonstrate the efficacy of detecting differential expression of genomic regions, a level of analysis not embraced by conventional workflows. Finally, we designed and conducted an experiment to identify hypothermia-responsive genes in terms of monotonic time-response. As a novel insight, hypothermia-dependent up-regulation of multiple genes of two major antioxidant pathways was identified and verified by quantitative real-time PCR.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Hipoxia de la Célula , Frío , Simulación por Computador , Silenciador del Gen , Ratones , Análisis de Secuencia de ARNRESUMEN
OBJECTIVE: Glucagon-like peptide 1 (GLP-1) receptor agonists are a new group of antidiabetic medications quickly gaining popularity. We aimed to examine behavioural and neuroendocrine changes following chronic treatment with GLP-1 receptor agonists in animal models. METHODS: The effects of chronic treatment with GLP-1 receptor agonists were determined on behavioural parameters [anxiety level in the light-dark compartment test, the motor activity in automated activity cages, immobility in the forced swimming test (FST)] and on corticosterone release in mice. The possible antidepressant effect of chronic liraglutide treatment was also studied in Flinders Sensitive Line (FSL) rats, a genetic model of depression. RESULTS: Two weeks of treatment with exenatide (10 µg/kg twice daily) or liraglutide (1200 µg/kg once daily) did not affect the anxiety level in a light-dark compartment test nor induce an antidepressant-like effect in the FST in mice. Moreover, chronic treatment with liraglutide had no effect on depression-related behaviour in FSL rats. Interestingly, hypolocomotion induced by the drugs in mice disappeared after chronic dosing. Both of the GLP-1 receptor agonists induced robust increases in corticosterone levels in mice under basal conditions as well as in the case of combination with swimming stress. Remarkably, exenatide was as potent a stimulator of corticosterone release after 2 weeks as after acute administration. CONCLUSIONS: The increases in corticosterone release seen after acute exenatide or liraglutide treatment do not abate after 2 weeks of treatment demonstrating that tolerance does not develop towards this particular effect of GLP-1 agonists.
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Corticosterona/metabolismo , Péptido 1 Similar al Glucagón/análogos & derivados , Péptidos/farmacología , Receptores de Glucagón/agonistas , Ponzoñas/farmacología , Animales , Conducta Animal/efectos de los fármacos , Glucemia/efectos de los fármacos , Corticosterona/sangre , Tolerancia a Medicamentos , Exenatida , Péptido 1 Similar al Glucagón/administración & dosificación , Péptido 1 Similar al Glucagón/farmacología , Receptor del Péptido 1 Similar al Glucagón , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacología , Liraglutida , Masculino , Ratones , Péptidos/administración & dosificación , Ratas , Ponzoñas/administración & dosificaciónAsunto(s)
Hormona Liberadora de Corticotropina/genética , Polimorfismo de Nucleótido Simple , Proopiomelanocortina/genética , Psoriasis/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proteína de Señalización Agouti/genética , Proteína Relacionada con Agouti/genética , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Oxidorreductasas Intramoleculares/genética , Masculino , Persona de Mediana Edad , Monofenol Monooxigenasa/genética , Fenotipo , Psoriasis/diagnóstico , Receptores de Melanocortina/genética , Factores de Riesgo , Adulto JovenRESUMEN
Immune regulation of the skin plays an important role in susceptibility and development of illnesses. The aim of our study was to localise the interleukin (IL)-10 family of cytokines, in children's skin and to determine possible age-related differences in the expression level. The mRNA expression level of IL10, IL19, IL20, IL22, IL24, IL26, IL28B, IL29 and their receptors IL10RA, IL10RB, IL20RA, IL20RB, IL22RA1, IL22RA2, IL28RA was compared in skin biopsies of children and adults and in childrens' skin cells by quantitative real-time PCR (qRT-PCR). Immunohistochemistry was performed to confirm the qRT-PCR findings. We found age-related differences in the expression of IL10RB, IL20, IL20RA, IL22RA1, IL22RA2, IL26 and IL28RA genes. Cell type-dependent expression of IL10 family cytokines was apparent in the skin. In addition to previously known differences in systemic immunological response of adults and children, the present results reveal differences in immune profile of adult and juvenile skin.
Asunto(s)
Interleucinas/metabolismo , Receptores de Interleucina/metabolismo , Piel/inmunología , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Biopsia , Células Cultivadas , Niño , Preescolar , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Lactante , Interleucinas/genética , Persona de Mediana Edad , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Interleucina/genética , Adulto JovenRESUMEN
BACKGROUND: Chemical fixation of the brain can be executed through either the immersion method or the perfusion method. Perfusion fixation allows for better preservation of the brain tissue's ultrastructure, as it provides rapid and uniform delivery of the fixative to the tissue. Still, not all facilities have the expertise to perform perfusion fixation, with initial high cost and complexity of perfusion systems as the main factors limiting its widespread usage. NEW METHOD: Here we present our low-cost approach of whole brain ex situ perfusion fixation to overcome the aforementioned limitations. Our self-made perfusion system, constructed utilising commercially accessible and affordable medical resources alongside laboratory and everyday items, demonstrates the capability to generate superior histological stainings of brain tissue. The perfused tissue can be stored prior to proceeding with IHC for at least one year. RESULTS: Our method yielded high-quality results in histological stainings using both free-floating cryosections and paraffin-embedded tissue sections. The system is fully reusable and complies with the principles of sustainable management. COMPARISON WITH EXISTING METHODS: Our whole brain perfusion system has been assembled from simple components and is able to achieve a linear flow with a pressure of 70 mmHg corresponding to the perfusion pressure of the brain. CONCLUSIONS: Our ex situ method can be especially useful in research settings where expensive perfusion systems are not affordable or in any field with high time pressure, making it suitable for the field of forensic medicine or pathology in general.
Asunto(s)
Encéfalo , Humanos , Inmunohistoquímica , Análisis Costo-Beneficio , Perfusión/métodos , Fijadores , Encéfalo/patologíaRESUMEN
Wolfram syndrome is a rare genetic disease caused by mutations in the WFS1 or CISD2 gene. A primary defect in Wolfram syndrome involves poor ER Ca2+ handling, but how this disturbance leads to the disease is not known. The current study, performed in primary neurons, the most affected and disease-relevant cells, involving both Wolfram syndrome genes, explains how the disturbed ER Ca2+ handling compromises mitochondrial function and affects neuronal health. Loss of ER Ca2+ content and impaired ER-mitochondrial contact sites in the WFS1- or CISD2-deficient neurons is associated with lower IP3R-mediated Ca2+ transfer from ER to mitochondria and decreased mitochondrial Ca2+ uptake. In turn, reduced mitochondrial Ca2+ content inhibits mitochondrial ATP production leading to an increased NADH/NAD+ ratio. The resulting bioenergetic deficit and reductive stress compromise the health of the neurons. Our work also identifies pharmacological targets and compounds that restore Ca2+ homeostasis, enhance mitochondrial function and improve neuronal health.
Asunto(s)
Calcio , Retículo Endoplásmico , Proteínas de la Membrana , Mitocondrias , Neuronas , Síndrome de Wolfram , Síndrome de Wolfram/metabolismo , Síndrome de Wolfram/genética , Calcio/metabolismo , Mitocondrias/metabolismo , Retículo Endoplásmico/metabolismo , Animales , Neuronas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones , Humanos , Adenosina Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Ratones Noqueados , NAD/metabolismo , Señalización del CalcioRESUMEN
The gene WFS1 encodes a protein with unknown function although its functional deficiency causes different neuropsychiatric and neuroendocrine syndromes. In the present study, we aimed to find the functional networks influenced by the time-dependent silencing of WFS1 in HEK cells. We performed whole genome gene expression profiling (Human Gene 1.0 ST Arrays) in HEK cells 24, 48, 72, and 96 h after transfection with three different WFS1 siRNAs. To verify silencing we performed quantitative RT-PCR and Western blot analysis. Analysis was conducted in two ways. First we analyzed the overall effect of the siRNA treatment on the gene expression profile. As a next step we performed time-course analysis separately for different siRNAs and combined for all siRNAs. Quantitative RT-PCR and Western blot analysis confirmed clear silencing of the expression of WFS1 after 48 h. Significant (FDR value<10%) changes in the expression of 11 genes was identified with most of these genes being related to the mitochondrial dysfunction and apoptosis. Time-course analysis confirmed significant correlations between WFS1 silencing and changes in the expression profiles of several genes. The pathways that were influenced significantly by WFS1 silencing were related to mitochondrial damage and neurodegenerative diseases. Our findings suggest a role of WFS1 gene in cell survival and its involvement in degenerative diseases.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Neurodegenerativas/metabolismo , Animales , Western Blotting , Línea Celular , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Modelos Teóricos , Enfermedades Neurodegenerativas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Síndrome de Wolfram/metabolismoRESUMEN
In mice, the limbic system-associated membrane protein (Lsamp) gene has been implicated in locomotion, anxiety, fear reaction, learning, social behaviour and adaptation. Human data links the LSAMP gene to several psychiatric disorders and completed suicide. Here, we investigated changes in major monoamine systems in mice lacking the Lsamp gene. First, the locomotor and rewarding effects of amphetamine were studied in Lsamp(-/-) mice and Lsamp(+/+) mice. Second, monoamine levels in major brain regions in response to saline and amphetamine injections were measured and, third, the expression levels of dopamine system-related genes in the brain were studied in these mice. Lsamp(-/-) mice displayed lower sensitivity to amphetamine in the motility box. Likewise, in the place preference test, the rewarding effect of amphetamine was absent in Lsamp(-/-) mice. In all brain regions studied, Lsamp(-/-) mice displayed lower serotonin (5-HT) baseline levels, but a greater 5-HT turnover rate, and amphetamine increased the level of 5-HT and lowered 5-HT turnover to a greater extent in Lsamp(-/-) mice. Finally, Lsamp(-/-) mice had lower level of dopamine transporter (DAT) mRNA in the mesencephalon. In conclusion, Lsamp-deficiency leads to increased endogenous 5-HT-ergic tone and enhanced 5-HT release in response to amphetamine. Elevated 5-HT function and reduced activity of DAT are the probable reasons for the blunted effects of amphetamine in these mice. Lsamp(-/-) mice are a promising model to study the neurobiological mechanisms of deviant social behaviour and adaptation impairment observed in many psychiatric disorders.
Asunto(s)
Anfetamina/farmacología , Moléculas de Adhesión Celular Neuronal/genética , Modelos Animales de Enfermedad , Trastornos Mentales/genética , Ratones , Serotonina/metabolismo , Trastorno de la Conducta Social/genética , Animales , Condicionamiento Psicológico , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Resistencia a Medicamentos/genética , Proteínas Ligadas a GPI/genética , Expresión Génica , Trastornos Mentales/psicología , Mesencéfalo/metabolismo , Ratones Noqueados , Actividad Motora , Recompensa , Trastorno de la Conducta Social/psicología , Lóbulo Temporal/metabolismoRESUMEN
The corticotrophin-releasing hormone-proopiomelanocortin (CRH-POMC) system in the skin coordinates pigmentation and the immune response. The aim of this study was to evaluate the regulatory role of the neuroendocrine system in the pathogenesis of psoriasis. Using quantitative real-time-PCR, mRNA expression levels of 15 genes related to the CRH-POMC system were measured in punch biopsies from lesional and non-lesional skin of patients with psoriasis and from skin of healthy control subjects. Statistically significant up-regulation of POMC, CRH receptor type 1, melanin-concentrating hormone receptor (MCHR1) and melanocortin receptors 2, 3 and 4 mRNA expression in lesional and in non-lesional skin compared with healthy control samples were established. Tyrosinase (TYR), T(Y)RP-1 and ASIP genes were statistically significantly down-regulated in lesional and non-lesional skin of psoriasis samples compared with healthy subjects. The up-regulation of POMC, melanocortin receptors, CRH receptor type 1 and MCHR1 in the lesional and non-lesional skin of psoriasis patients supports the importance of the local CRH-POMC system in the pathogenesis of psoriasis.
Asunto(s)
Hormona Liberadora de Corticotropina/genética , Perfilación de la Expresión Génica , Proopiomelanocortina/genética , Psoriasis/genética , ARN Mensajero/análisis , Transducción de Señal/genética , Piel/química , Adolescente , Adulto , Proteína de Señalización Agouti/genética , Biopsia , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica/métodos , Predisposición Genética a la Enfermedad , Humanos , Masculino , Glicoproteínas de Membrana/genética , Persona de Mediana Edad , Monofenol Monooxigenasa/genética , Oxidorreductasas/genética , Fenotipo , Psoriasis/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Melanocortina/genética , Receptores de Somatostatina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la Enfermedad , Piel/patología , Adulto JovenRESUMEN
Physical characteristics of the growth substrate including nano- and microstructure play crucial role in determining the behaviour of the cells in a given biological context. To test the effect of varying the supporting surface structure on cell growth we applied a novel sol-gel phase separation-based method to prepare micro- and nanopatterned surfaces with round surface structure features. Variation in the size of structural elements was achieved by solvent variation and adjustment of sol concentration. Growth characteristics and morphology of primary human dermal fibroblasts were found to be significantly modulated by the microstructure of the substrate. The increase in the size of the structural elements, lead to increased inhibition of cell growth, altered morphology (increased cytoplasmic volume), enlarged cell shape, decrease in the number of filopodia) and enhancement of cell senescence. These effects are likely mediated by the decreased contact between the cell membrane and the growth substrate. However, in the case of large surface structural elements other factors like changes in the 3D topology of the cell's cytoplasm might also play a role.