Gerburg Keilhoff

2,7-Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation

Reactive oxygen species (ROS) have been implicated as an important causative factor in cell damage, including apoptosis and necrosis. Their proposed actions comprise lipid peroxidation, DNA damage, destruction of the mitochondrial respiratory chain and protein modifications. Recent experiments underline the importance of peroxynitrite, the reaction product of the two potent reactive species nitric oxide and superoxide. Several fluorogenic compounds have been used in order to determine ROS formation in living cells. Besides dihydrorhodamine‐123 (DHR‐123), at present mostly applied to monitor peroxynitrite, 2,7‐dihydrodichlorofluorescein (DCF‐H) is used for detection of hydrogen peroxide and nitric oxide. We employed a cell free approach to evaluate the specificity and sensitivity of DCF‐H to various oxidizing compounds. Our studies imply that DCF‐H is much more sensitive to peroxynitrite oxidation than any other compound tested. In order to study peroxynitrite generation within individual cells, primary glial cultures loaded with DCF‐H were monitored with a laser scanning microscope. Microglia, stimulated to simultaneously produce the peroxynitrite precursors nitric oxide and superoxide, displayed the greatest increase in DCF fluorescence, whereas microglia producing either nitric oxide or superoxide alone showed a relatively small increase in DCF fluorescence. In conclusion, DCF‐H was demonstrated to be an excellent peroxynitrite marker with the potential to detect peroxynitrite formation in living cells.

Evidence for a wide extra-astrocytic distribution of S100B in human brain

BackgroundS100B is considered an astrocytic in-situ marker and protein levels in cerebrospinal fluid (CSF) or serum are often used as biomarker for astrocytic damage or dysfunction. However, studies on S100B in the human brain are rare. Thus, the distribution of S100B was studied by immunohistochemistry in adult human brains to evaluate its cell-type specificity.ResultsContrary to glial fibrillary acidic protein (GFAP), which selectively labels astrocytes and shows only faint ependymal immunopositivity, a less uniform staining pattern was seen in the case of S100B. Cells with astrocytic morphology were primarily stained by S100B in the human cortex, while only 20% (14–30%) or 14% (7–35%) of all immunopositive cells showed oligodendrocytic morphology in the dorsolateral prefrontal and temporal cortices, respectively. In the white matter, however, most immunostained cells resembled oligodendrocytes [frontal: 75% (57–85%); temporal: 73% (59–87%); parietal: 79% (62–89%); corpus callosum: 93% (86–97%)]. S100B was also found in ependymal cells, the choroid plexus epithelium, vascular endothelial cells, lymphocytes, and several neurones. Anti-myelin basic protein (MBP) immunolabelling showed an association of S100B with myelinated fibres, whereas GFAP double staining revealed a distinct subpopulation of cells with astrocytic morphology, which solely expressed S100B but not GFAP. Some of these cells showed co-localization of S100B and A2B5 and may be characterized as O2A glial progenitor cells. However, S100B was not detected in microglial cells, as revealed by double-immunolabelling with HLA-DR.ConclusionS100B is localized in many neural cell-types and is less astrocyte-specific than GFAP. These are important results in order to avoid misinterpretation in the identification of normal and pathological cell types in situ and in clinical studies since S100B is continuously used as an astrocytic marker in animal models and various human diseases.

Repeated application of ketamine to rats induces changes in the hippocampal expression of parvalbumin, neuronal nitric oxide synthase and cFOS similar to those found in human schizophrenia

Treatment with the phencyclidine derivative ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist and a well known anesthetic, has recently been introduced to mimic schizophrenia in animals. Using rats repeatedly treated with sub-anesthetic doses we demonstrate in the hippocampal formation the cellular distribution patterns of proteins being relevant to the pathogenesis of schizophrenia. Compared with controls an increase in the density of reduced nicotinamide adenine dinucleotide phosphate diaphorase-, neuronal nitric oxide synthase- and cFOS-positive hippocampal interneurons was found, whereas the density of parvalbumin expressing cells was decreased. Our experiments show that repeated injections of sub-anesthetic doses of ketamine induce significant changes in the nitrergic and GABAergic system which, in part, resemble those described in postmortem brains of human schizophrenics indicating that sub-chronic treatment with sub-anesthetic doses of ketamine might be a useful animal model to study schizophrenia.

The many faces of nitric oxide in schizophrenia. A review

Intense research has been conducted in an effort to identify specific biological markers of schizophrenia. The gas nitric oxide (NO) is one of the most important signaling molecules involved in a plethora of cellular events that take place in the cardiovascular, immune and nervous systems of animals. This survey aims to demonstrate that NO and its metabolites play important roles in schizophrenia and have a significant influence on our understanding of the development, progression and treatment of the disease. Special emphasis is given to the impact of NO metabolism on processes known to be disturbed in schizophrenia (i.e., cell migration, formation of synapses, NMDA receptor mediated neurotransmission, membrane pathology and cognitive abilities). However, when comparing data on the NO metabolism in the brain tissue and body fluids of schizophrenics with those obtained from patients with other neurological and psychiatric diseases, it becomes clear that alterations of NO metabolism are not unique to, or indicative of, schizophrenia. Thus, NO and its metabolites are not suitable diagnostic tools to distinguish schizophrenia from psychically healthy control cases or from other brain disorders.

Immunohistochemical evidence for impaired neuregulin-1 signaling in the prefrontal cortex in schizophrenia and in unipolar depression.

Abstract:  In the central nervous system (CNS), neuregulin‐1 (NRG‐1) proteins function in neuronal migration, differentiation, and survival of oligodendrocytes. The NRG‐1 gene codes for at least 15 different isoforms, which may be classified on the basis of their molecular structure. At least two different haplotypes of the NRG‐1 gene may be associated with schizophrenia. An abnormal expression pattern of NRG‐1 mRNA was found in the prefrontal cortex of schizophrenic patients in comparison to controls. We here show that the NRG‐1α isoform is significantly reduced in white matter of the prefrontal cortex in schizophrenia but not in affective disorder. In the prefrontal gray matter, the density of NRG‐1α expressing neurons was reduced in individuals with schizophrenia and in unipolar patients. We studied brains of 22 schizophrenics, 12 patients with affective disorders (7 unipolar and 5 bipolar), and 22 matched controls. NRG‐1α immunoreactive material was detected with a polyclonal antiserum against the synthetic peptide from α‐type EGF‐like domain of human NRG. The demonstrated decreased number of NRG‐1 immunoreactive neurons in the brains of schizophrenics and patients with unipolar depression points to an important role of this NRG‐1α splice variant in neuropsychiatric disorders. Reduced NRG‐1α protein concentrations were found in brains of schizophrenics after Western blot analysis. The diminished expression of NRG‐1α strongly supports an early neurodevelopmental component to schizophrenia.

Cell Proliferation is Influenced by Bulbectomy and Normalized by Imipramine Treatment in a Region-Specific Manner

Growing evidence indicates that alterations of neuroplasticity may contribute to the pathophysiology of depression. In contrast, various antidepressants increase adult hippocampal neurogenesis and block the effects of stress. These findings result in the ‘neurogenesis hypothesis of depression’. The present study seeks to determine out whether cell proliferation is altered in the hippocampus, subventricular zone (SVZ), and basolateral amygdala of adult rats exposed to bilateral olfactory bulbectomy, another established model of depression and, if so, how imipramine effects bulbectomy-induced changes of cell genesis. Bulbectomy results in a significant reduction of cell proliferation in the hippocampus and SVZ, an effect that is normalized by subchronic doses of imipramine. Moreover, an increase in cell genesis in the basolateral amygdala, which is not affected by imipramine, is demonstrated. TUNEL staining indicates an enhanced apoptosis after bulbectomy in the SVZ that cannot be reduced by imipramine. Cell death rates in the hippocampus and amygdala are not affected by bulbectomy. The opposing effects of bulbectomy and imipramine treatment in the hippocampus and amygdala demonstrate that these structures of the limbic system, both integrated in emotional processing, react quite differently with regard to neuroplasticity. Further to this, we discuss a possible link between the pathogenesis of depression and changed neuronal plasticity in the SVZ.

Patterns of nitric oxide synthase at the messenger RNA and protein levels during early rat brain development

There is substantial evidence that the intra- and intercellular messenger nitric oxide, generated enzymatically from L-arginine by nitric oxide synthase in different isoforms, is involved in the development of nervous tissue. In this study we investigated the nitric oxide expression in the pre- and postnatally developing rat brain. With regard to messenger RNA, all of the basic nitric oxide synthase isoforms (neuronal, endothelial and macrophage nitric oxide synthase) were already expressed at embryonic day 10 and showed a temporary decrease at embryonic day 17. Western blot analysis of the three isoform proteins revealed a time pattern that was different from those of messenger RNAs. Although the endothelial nitric oxide synthase isoform was also expressed at embryonic day 10, no quantitative changes were observed over the whole time period studied. Protein amounts of brain and inducible nitric oxide synthase were first detectable at embryonic day 15, with a tendency to rise. A parallel time pattern was found for the NADPH-diaphorase activity in our light microscopic studies, whereas ultrastructurally the reaction product was seen in the brain pallium even of 13-day-old embryos. The data indicate a permanent presence of the transcripts for all nitric oxide synthase isoforms in the rat central nervous system from embryonic day 10 onwards, although the expression of respective proteins and staining patterns may differ.

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

BackgroundCardiac 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.Therefore, the present study aimed to determine the expression stability of ten commonly used reference genes (Actb, actin, beta; B2m, beta-2 microglobulin;CypA, cyclophilin A; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Hprt, hypoxanthine guanine phosphoribosyl transferase; Pgk1, phosphoglycerate kinase 1; Rpl13a, ribosomal protein L13A; Sdha, succinat dehydrogenase complex, subunit a, flavoprotein (Fp); Tbp, TATA box binding protein; Ywhaz, 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.ResultsThe microglial marker Mac-1, used as a target gene to validate the experimental model, was found to be upregulated about 10- to 20-fold after cardiac arrest.Expression stability of candidate reference genes was analyzed using geNorm and NormFinder software tools. Several of these genes behave rather stable. CypA and Pgk1 were identified by geNorm as the two most stable genes 4 and 21 days after asphyxial cardiac arrest, CypA and Gapdh at 7 days post treatment. B2m turned out to be the most variable candidate reference gene, being about 2-fold upregulated in the cardiac arrest treatment groups.ConclusionWe 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 CypA and Pgk1 should be considered 4 and 21 days post injury, whereas CypA and Gapdh is the best combination at 7 days. CypA is most favorable if restriction to a single reference gene for all time points is required.

Distinct Ca2+ thresholds determine cytochrome c release or permeability transition pore opening in brain mitochondria

In diseases associated with neuronal degeneration, such as Alzheimer’s or cerebral ischemia, the cytosolic Ca2+ concentration ([Ca2+]cyt) is pathologically elevated. It is still unclear, however, under which conditions Ca2+ induces either apoptotic or necrotic neuronal cell death. Studying respiration and morphology of rat brain mitochondria, we found that extramitochondrial [Ca2+] above 1 μM causes reversible release of cytochrome c, a key trigger of apoptosis. This event was NO‐independent but required Ca2+ influx into the mitochondrial matrix. The mitochondrial permeability transition pore (PTP), widely thought to underlie cytochrome c release, was not involved. In contrast to noncerebral tissue, only relatively high [Ca2+] (≈ 200 μM) opened PTP and ruptured mitochondria. Our findings might reflect a fundamental mechanism to protect postmitotic neuronal tissue against necrotic devastation and inflammation.

Distribution, Targeting, and Internalization of the sst4 Somatostatin Receptor in Rat Brain

Somatostatin mediates its diverse physiological effects through a family of five G-protein-coupled receptors (sst1–sst5); however, knowledge about the distribution of individual somatostatin receptor proteins in mammalian brain is incomplete. In the present study, we have examined the regional and subcellular distribution of the somatostatin receptor sst4 in the rat CNS by raising anti-peptide antisera to the C-terminal tail of sst4. The specificity of affinity-purified antibodies was demonstrated using immunofluorescent staining of HEK 293 cells stably transfected with an epitope-tagged sst4 receptor. In Western blotting, the antiserum reacted specifically with a broad band in rat brain, which migrated at ∼70 kDa before and ∼50 kDa after enzymatic deglycosylation. sst4-Like immunoreactivity was most prominent in many forebrain regions, including the cerebral cortex, hippocampus, striatum, amygdala, and hypothalamus. Analysis at the electron microscopic level revealed that sst4-expressing neurons target this receptor preferentially to their somatodendritic domain. Like the sst2A receptor, sst4-immunoreactive dendrites were often closely apposed by somatostatin-14-containing fibers and terminals. However, unlike the sst2A receptor, sst4 was not internalized in response to intracerebroventricular administration of somatostatin-14. After percussion trauma of the cortex, neuronal sst4 receptors progressively declined at the sites of damage. This decline coincided with an induction of sst4 expression in cells with a glial-like morphology. Together, this study provides the first description of the distribution of immunoreactive sst4receptor proteins in rat brain. We show that sst4 is strictly somatodendritic and most likely functions in a postsynaptic manner. In addition, the sst4 receptor may have a previously unappreciated function during the neuronal degeneration–regeneration process.