Histamine release after intravenous application of short-acting hypnotics. A comparison of etomidate, Althesin (CT1341) and propanidid

The subject of histamine release was investigated in 16 volunteers by means of plasma histamine determination after the administration of etornidate, Althesin, propanidid, and Cremophor EL. Althesin and propanidid caused release of histamine in various degrees of frequency. Blood pressure changes were rather pronounced with both anaesthetic agents; tachycardia reached its maximum in the first and second minute, which seems to be an argument against histamine release as the underlying cause of this reaction. Histamine was, indeed, only released to such an extent (with the exception of one borderline case) that no clinical symptoms other than secretion of gastric juice and erythema were to be expected. After the application of etomidate and Cremophor EL an increase in plasma histamine was not detectable. Changes in the differential blood picture in terms of a decrease in basophils only occurred after Althesin and propanidid; not, however, after etomidate and Cremophor EL. Etomidate is, therefore, the first hypnotic drug for intravenous application which is unlikely to cause chemical histamine release

Expression of Kv3.1b potassium channel is widespread in macaque motor cortex pyramidal cells: A histological comparison between rat and macaque

There are substantial differences across species in the organisation and function of the motor pathways. These differences extend to basic electrophysiological properties. Thus, in rat motor cortex, pyramidal cells have long duration action potentials, while in the macaque, some pyramidal neurons exhibit short duration 'thin' spikes. These differences may be related to the expression of the fast potassium channel Kv3.1b, which in rat interneurons is associated with generation of thin spikes. Rat pyramidal cells typically lack these channels, while there are reports that they are present in macaque pyramids. Here we made a systematic, quantitative comparison of the expression of Kv3.1b in sections from macaque and rat motor cortex, using two different antibodies (NeuroMab, Millipore). As our standard reference, we examined, in the same sections, Kv3.1b staining in parvalbumin-positive interneurons, which show strong Kv3.1b immunoreactivity. In macaque motor cortex, a large sample of pyramidal neurons were nearly all found to express Kv3.1b in their soma membranes. These labelled neurons were identified as pyramidal based either by expression of SMI32 (a pyramidal marker), or by their shape and size, lack of expression of parvalbumin (a marker for some classes of interneuron). Large (Betz cells), medium and small pyramidal neurons all expressed Kv3.1b. In rat motor cortex, SMI32-postive pyramidal neurons expressing Kv3.1b were very rare and weakly stained. Thus, there is a marked species difference in the immunoreactivity of Kv3.1b in pyramidal neurons, and this may be one of the factors explaining the pronounced electrophysiological differences between rat and macaque pyramidal neurons

Hereditary sensory neuropathy type 1-associated deoxysphingolipids cause neurotoxicity, acute calcium handling abnormalities and mitochondrial dysfunction in vitro

Hereditary sensory neuropathy type 1 (HSN-1) is a peripheral neuropathy most frequently caused by mutations in the SPTLC1 or SPTLC2 genes, which code for two subunits of the enzyme serine palmitoyltransferase (SPT). SPT catalyzes the first step of de novo sphingolipid synthesis. Mutations in SPT result in a change in enzyme substrate specificity, which causes the production of atypical deoxysphinganine and deoxymethylsphinganine, rather than the normal enzyme product, sphinganine. Levels of these abnormal compounds are elevated in blood of HSN-1 patients and this is thought to cause the peripheral motor and sensory nerve damage that is characteristic of the disease, by a largely unresolved mechanism. In this study, we show that exogenous application of these deoxysphingoid bases causes dose- and time-dependent neurotoxicity in primary mammalian neurons, as determined by analysis of cell survival and neurite length. Acutely, deoxysphingoid base neurotoxicity manifests in abnormal Ca2+ handling by the endoplasmic reticulum (ER) and mitochondria as well as dysregulation of cell membrane store-operated Ca2+ channels. The changes in intracellular Ca2+ handling are accompanied by an early loss of mitochondrial membrane potential in deoxysphingoid base-treated motor and sensory neurons. Thus, these results suggest that exogenous deoxysphingoid base application causes neuronal mitochondrial dysfunction and Ca2+ handling deficits, which may play a critical role in the pathogenesis of HSN-1

Enhanced Expression of TRAP1 Protects Mitochondrial Function in Motor Neurons under Conditions of Oxidative Stress

TNF‐receptor associated protein (TRAP1) is a cytoprotective mitochondrial‐specific member of the Hsp90 heat shock protein family of protein chaperones that has been shown to antagonise mitochondrial apoptosis and oxidative stress, regulate the mitochondrial permeability transition pore and control protein folding in mitochondria. Here we show that overexpression of TRAP1 protects motor neurons from mitochondrial dysfunction and death induced by exposure to oxidative stress conditions modelling amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease in which motor neurons degenerate, leading to muscle weakness and atrophy and death, typically within 3 years of diagnosis. In primary murine motor neurons, shRNAmediated knockdown of TRAP1 expression results in mitochondrial dysfunction but does not further exacerbate damage induced by oxidative stress alone. Together, these results show that TRAP1 may be a potential therapeutic target for neurodegenerative diseases such as ALS, where mitochondrial dysfunction has been shown to be an early marker of pathogenesis

Bi-allelic JAM2 Variants Lead to Early-Onset Recessive Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder characterized by a combination of neurological, psychiatric, and cognitive decline associated with calcium deposition on brain imaging. To date, mutations in five genes have been linked to PFBC. However, more than 50% of individuals affected by PFBC have no molecular diagnosis. We report four unrelated families presenting with initial learning difficulties and seizures and later psychiatric symptoms, cerebellar ataxia, extrapyramidal signs, and extensive calcifications on brain imaging. Through a combination of homozygosity mapping and exome sequencing, we mapped this phenotype to chromosome 21q21.3 and identified bi-allelic variants in JAM2. JAM2 encodes for the junctional-adhesion-molecule-2, a key tight-junction protein in blood-brain-barrier permeability. We show that JAM2 variants lead to reduction of JAM2 mRNA expression and absence of JAM2 protein in patient's fibroblasts, consistent with a loss-of-function mechanism. We show that the human phenotype is replicated in the jam2 complete knockout mouse (jam2 KO). Furthermore, neuropathology of jam2 KO mouse showed prominent vacuolation in the cerebral cortex, thalamus, and cerebellum and particularly widespread vacuolation in the midbrain with reactive astrogliosis and neuronal density reduction. The regions of the human brain affected on neuroimaging are similar to the affected brain areas in the myorg PFBC null mouse. Along with JAM3 and OCLN, JAM2 is the third tight-junction gene in which bi-allelic variants are associated with brain calcification, suggesting that defective cell-to-cell adhesion and dysfunction of the movement of solutes through the paracellular spaces in the neurovascular unit is a key mechanism in CNS calcification

Regional Differences in Heat Shock Protein 25 Expression in Brain and Spinal Cord Astrocytes of Wild-Type and SOD1 (G93A) Mice

Heterogeneity of glia in different CNS regions may contribute to the selective vulnerability of neuronal populations in neurodegenerative conditions such as amyotrophic lateral sclerosis (ALS). Here, we explored regional variations in the expression of heat shock protein 25 in glia under conditions of acute and chronic stress. Hsp27 (Hsp27; murine orthologue: Hsp25) fulfils a number of cytoprotective functions and may therefore be a possible therapeutic target in ALS. We identified a subpopulation of astrocytes in primary murine mixed glial cultures that expressed Hsp25. Under basal conditions, the proportion of Hsp25-positive astrocytes was twice as high in spinal cord cultures than in cortical cultures. To explore the physiological role of the elevated Hsp25 expression in spinal cord astrocytes, we exposed cortical and spinal cord glia to acute stress, using heat stress and pro-inflammatory stimuli. Surprisingly, we observed no stress-induced increase in Hsp25 expression in either cortical or spinal cord astrocytes. Similarly, exposure to endogenous stress, as modelled in glial cultures from SOD1 G93A-ALS mice, did not increase Hsp25 expression above that observed in astrocytes from wild-type mice. In vivo, Hsp25 expression was greater under conditions of chronic stress present in the spinal cord of SOD1 G93A mice than in wild-type mice, although this increase in expression is likely to be due to the extensive gliosis that occurs in this model. Together, these results show that there are differences in the expression of Hsp25 in astrocytes in different regions of the central nervous system, but Hsp25 expression is not upregulated under acute or chronic stress conditions

Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

Background: Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). Methods: Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. Results: Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. Conclusions: Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material

Sectioning and Counting of Motor Neurons in the L3 to L6 Region of the Adult Mouse Spinal Cord

Histology is the study of the microscopic structure of tissues. This protocol permits the generation of frozen transverse sections of lumbar spinal cord regions L3 to L6. It enables counting of murine ventral horn lumbar motor neurons in a reproducible manner. Methods include spinal column dissection, hydraulic extrusion, and histological processing. The preparation for cryo-sectioning includes embedding lumbar spinal cord in optimal cutting temperature (OCT) medium. The correct orientation of the tissue is critical as calculating the amount of tissue to discard saved time overall. Specific details regarding section thickness and mounting are described. These requirements not only allow optimum coverage of specific regions but also ensure that no individual motor neuron was counted twice. The Nissl bodies of the motor neurons were stained using gallocyanin. The sections obtained are all of a comparable area and quality assurance is consistent. The specificity of the staining enables the scientist to identify and reliably quantify lumbar motor neurons. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Euthanasia of mouse and isolation of spinal cord. Basic Protocol 2: Hydraulic extrusion of the spinal cord. Basic Protocol 3: Identification of the lumbar region. Basic Protocol 4: Embedding cord in OCT. Basic Protocol 5: Collection of frozen sections onto slides. Basic Protocol 6: Gallocyanin staining. Basic Protocol 7: Motor neuron counting

Modeling Stem Cell Induction Processes

Technology for converting human cells to pluripotent stem cell using induction processes has the potential to revolutionize regenerative medicine. However, the production of these so called iPS cells is still quite inefficient and may be dominated by stochastic effects. In this work we build mass-action models of the core regulatory elements controlling stem cell induction and maintenance. The models include not only the network of transcription factors NANOG, OCT4, SOX2, but also important epigenetic regulatory features of DNA methylation and histone modification. We show that the network topology reported in the literature is consistent with the observed experimental behavior of bistability and inducibility. Based on simulations of stem cell generation protocols, and in particular focusing on changes in epigenetic cellular states, we show that cooperative and independent reaction mechanisms have experimentally identifiable differences in the dynamics of reprogramming, and we analyze such differences and their biological basis. It had been argued that stochastic and elite models of stem cell generation represent distinct fundamental mechanisms. Work presented here suggests an alternative possibility that they represent differences in the amount of information we have about the distribution of cellular states before and during reprogramming protocols. We show further that unpredictability and variation in reprogramming decreases as the cell progresses along the induction process, and that identifiable groups of cells with elite-seeming behavior can come about by a stochastic process. Finally we show how different mechanisms and kinetic properties impact the prospects of improving the efficiency of iPS cell generation protocols.Fundação para a Ciência e a Tecnologia (BD 42942)MIT-Portugal ProgramNational Institutes of Health (U.S.) (CA112967)Singapore–MIT Alliance for Research and TechnologyIntel Corporatio

Narcolepsy patients have antibodies that stain distinct cell populations in rat brain and influence sleep patterns.

Narcolepsy is a chronic sleep disorder, likely with an autoimmune component. During 2009 and 2010, a link between A(H1N1)pdm09 Pandemrix vaccination and onset of narcolepsy was suggested in Scandinavia. In this study, we searched for autoantibodies related to narcolepsy using a neuroanatomical array: rat brain sections were processed for immunohistochemistry/double labeling using patient sera/cerebrospinal fluid as primary antibodies. Sera from 89 narcoleptic patients, 52 patients with other sleep-related disorders (OSRDs), and 137 healthy controls were examined. Three distinct patterns of immunoreactivity were of particular interest: pattern A, hypothalamic melanin-concentrating hormone and proopiomelanocortin but not hypocretin/orexin neurons; pattern B, GABAergic cortical interneurons; and pattern C, mainly globus pallidus neurons. Altogether, 24 of 89 (27%) narcoleptics exhibited pattern A or B or C. None of the patterns were exclusive for narcolepsy but were also detected in the OSRD group at significantly lower numbers. Also, some healthy controls exhibited these patterns. The antigen of pattern A autoantibodies was identified as the common C-terminal epitope of neuropeptide glutamic acid-isoleucine/alpha-melanocyte-stimulating hormone (NEI/alphaMSH) peptides. Passive transfer experiments on rat showed significant effects of pattern A human IgGs on rapid eye movement and slow-wave sleep time parameters in the inactive phase and EEG theta-power in the active phase. We suggest that NEI/alphaMSH autoantibodies may interfere with the fine regulation of sleep, contributing to the complex pathogenesis of narcolepsy and OSRDs. Also, patterns B and C are potentially interesting, because recent data suggest a relevance of those brain regions/neuron populations in the regulation of sleep/arousal