Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model

<p>Abstract</p> <p>Background</p> <p>Cardiac arrest, and the associated arrest of blood circulation, immediately leads to permanent brain damage because of the exhaustion of oxygen, glucose and energy resources in the brain. Most hippocampal CA1 neurons die during the first week post the insult. Molecular data concerning the recovery after resuscitation are sparse and limited to the early time period. Expression analysis of marker genes via quantitative real-time RT-PCR enables to follow up the remodeling process. However, proper validation of the applied normalization strategy is a crucial prerequisite for reliable conclusions.</p> <p>Therefore, the present study aimed to determine the expression stability of ten commonly used reference genes (<it>Actb</it>, actin, beta; <it>B2m</it>, beta-2 microglobulin;<it>CypA</it>, cyclophilin A; <it>Gapdh</it>, glyceraldehyde-3-phosphate dehydrogenase; <it>Hprt</it>, hypoxanthine guanine phosphoribosyl transferase; <it>Pgk1</it>, phosphoglycerate kinase 1; <it>Rpl13a</it>, ribosomal protein L13A; <it>Sdha</it>, succinat dehydrogenase complex, subunit a, flavoprotein (Fp); <it>Tbp</it>, TATA box binding protein; <it>Ywhaz</it>, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) in the rat hippocampus four, seven and twenty-one days after cardiac arrest. Moreover, experimental groups treated with the anti-inflammatory and anti-apoptotic drug minocycline have been included in the study as well.</p> <p>Results</p> <p>The microglial marker <it>Mac-1</it>, used as a target gene to validate the experimental model, was found to be upregulated about 10- to 20-fold after cardiac arrest.</p> <p>Expression stability of candidate reference genes was analyzed using geNorm and NormFinder software tools. Several of these genes behave rather stable. <it>CypA </it>and <it>Pgk1 </it>were identified by geNorm as the two most stable genes 4 and 21 days after asphyxial cardiac arrest, <it>CypA </it>and <it>Gapdh </it>at 7 days post treatment. <it>B2m </it>turned out to be the most variable candidate reference gene, being about 2-fold upregulated in the cardiac arrest treatment groups.</p> <p>Conclusion</p> <p>We have validated endogenous control genes for qRT-PCR analysis of gene expression in rat hippocampus after resuscitation from cardiac arrest. For normalization purposes in gene profiling studies a combination of <it>CypA </it>and <it>Pgk1 </it>should be considered 4 and 21 days post injury, whereas <it>CypA </it>and <it>Gapdh </it>is the best combination at 7 days. <it>CypA </it>is most favorable if restriction to a single reference gene for all time points is required.</p

Magnetic nanoparticles in primary neural cell cultures are mainly taken up by microglia

<p>Abstract</p> <p>Background</p> <p>Magnetic nanoparticles (MNPs) offer a large range of applications in life sciences. Applications in neurosciences are one focus of interest. Unfortunately, not all groups have access to nanoparticles or the possibility to develop and produce them for their applications. Hence, they have to focus on commercially available particles. Little is known about the uptake of nanoparticles in primary cells. Previously studies mostly reported cellular uptake in cell lines. Here we present a systematic study on the uptake of magnetic nanoparticles (MNPs) by primary cells of the nervous system.</p> <p>Results</p> <p>We assessed the internalization in different cell types with confocal and electron microscopy. The analysis confirmed the uptake of MNPs in the cells, probably with endocytotic mechanisms. Furthermore, we compared the uptake in PC12 cells, a rat pheochromocytoma cell line, which is often used as a neuronal cell model, with primary neuronal cells. It was found that the percentage of PC12 cells loaded with MNPs was significantly higher than for neurons. Uptake studies in primary mixed neuronal/glial cultures revealed predominant uptake of MNPs by microglia and an increase in their number. The number of astroglia and oligodendroglia which incorporated MNPs was lower and stable. Primary mixed Schwann cell/fibroblast cultures showed similar MNP uptake of both cell types, but the Schwann cell number decreased after MNP incubation. Organotypic co-cultures of spinal cord slices and peripheral nerve grafts resembled the results of the dispersed primary cell cultures.</p> <p>Conclusions</p> <p>The commercial MNPs used activated microglial phagocytosis in both disperse and organotypic culture systems. It can be assumed that <it>in vivo </it>application would induce immune system reactivity, too. Because of this, their usefulness for <it>in vivo </it>neuroscientific implementations can be questioned. Future studies will need to overcome this issue with the use of cell-specific targeting strategies. Additionally, we found that PC12 cells took up significantly more MNPs than primary neurons. This difference indicates that PC12 cells are not a suitable model for natural neuronal uptake of nanoparticles and qualify previous results in PC12 cells.</p

Growth factor choice is critical for successful functionalization of nanoparticles

Nanoparticles (NPs) show new characteristics compared to the corresponding bulk material. These nanoscale properties make them interesting for various applications in biomedicine and life sciences. One field of application is the use of magnetic NPs to support regeneration in the nervous system. Drug delivery requires a functionalization of NPs with bio-functional molecules. In our study, we functionalized self-made PEI-coated iron oxide NPs with nerve growth factor (NGF) and glial cell-line derived neurotrophic factor (GDNF). Next, we tested the bio-functionality of NGF in a rat pheochromocytoma cell line (PC12) and the bio-functionality of GDNF in an organotypic spinal cord culture. Covalent binding of NGF to PEI-NPs impaired bio-functionality of NGF, but non-covalent approach differentiated PC12 cells reliably. Non-covalent binding of GDNF showed a satisfying bio-functionality of GDNF:PEI-NPs, but turned out to be unstable in conjugation to the PEI-NPs. Taken together, our study showed the importance of assessing bio-functionality and binding stability of functionalized growth factors using proper biological models. It also shows that successful functionalization of magnetic NPs with growth factors is dependent on the used binding chemistry and that it is hardly predictable. For use as therapeutics, functionalization strategies have to be reproducible and future studies are needed

NERVEN AUS DEM LABOR: DAS TISSUE ENGINEERING PERIPHERER NERVEN ZUR VERSORGUNG VON NERVENVERLETZUNGEN

Verletzungen peripherer Nerven sind häufiger als gemeinhin angenommen und werden in Deutschland noch nicht optimal behandelt. Die besten Vorraussetzungen für eine gute Rekonstruktion bietet die primäre mikrochirurgische Nervennaht. Bei Defekten wird ein körpereigenes Transplantat benötigt. Dessen Entnahme führt allerdings zu Funktionsausfällen. Unsere interdisziplinäre Forschungsgruppe aus Plastischen Chirurgen und Neurobiologen beschäftigt sich mit der labortechnischen Herstellung von Nervenersatzgewebe aus einer biogenen Trägermatrix und den nerveneigenen Hüllzellen (sog. Schwannsche Zellen), die die Regeneration der Nerven verbessern

Modelling the dopamine and noradrenergic cell loss that occurs in Parkinson's disease and the impact on hippocampal neurogenesis

Key pathological features of Parkinson's Disease (PD) include the progressive degeneration of midbrain dopaminergic (DA) neurons and hindbrain noradrenergic (NA) neurons. The loss of DA neurons has been extensively studied and is the main cause of motor dysfunction. Importantly, however, there are a range of ‘non‐movement’ related features of PD including cognitive dysfunction, sleep disturbances and mood disorders. The origins for these non‐motor symptoms are less clear, but a possible substrate for cognitive decline may be reduced adult‐hippocampal neurogenesis, which is reported to be impaired in PD. The mechanisms underlying reduced neurogenesis in PD are not well established. Here we tested the hypothesis that NA and DA depletion, as occurs in PD, impairs hippocampal neurogenesis. We used 6‐hydroxydopamine or the immunotoxin dopamine‐β‐hydroxylase‐saporin to selectively lesion DA or NA neurons, respectively, in adult Sprague Dawley rats and assessed hippocampal neurogenesis through phenotyping of cells birth‐dated using 5‐bromo‐2′‐deoxyuridine. The results showed no difference in proliferation or differentiation of newborn cells in the subgranular zone of the dentate gyrus after NA or DA lesions. This suggests that impairment of hippocampal neurogenesis in PD likely results from mechanisms independent of, or in addition to degeneration of DA and NA neurons

Alteration in the plasma concentration of a DAAO inhibitor, 3-methylpyrazole-5-carboxylic acid, in the ketamine-treated rats and the influence on the pharmacokinetics of plasma d-tryptophan

A determination method for 3-methylpyrazole-5-carboxylic acid (MPC), an inhibitor of d-amino acid oxidase (DAAO), in rat plasma was developed by using high-performance liquid chromatography-mass spectrometry (LC-MS). The structural isomer of MPC, 3-methylpyrazole-4-carboxylic acid, was used as an internal standard, and the intra- and inter-day accuracies and precisions were satisfactory for the determination of plasma MPC