immunohistochemistry-multiple-labeling-with-antibodies-from-same-host

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Jena University Hospital

Oxidation products of low-density lipoproteins have been suggested to promote inflammation during atherogenesis, and reticulocyte-type 15-lipoxygenase has been implicated to mediate this oxidation. In addition, the 5-lipoxygenase cascade leads to formation of leukotrienes, which exhibit strong proinflammatory activities in cardiovascular tissues. Here, we studied both lipoxygenase pathways in human atherosclerosis. The 5-lipoxygenase pathway was abundantly expressed in arterial walls of patients afflicted with various lesion stages of atherosclerosis of the aorta and of coronary and carotid arteries. 5-lipoxygenase localized to macrophages, dendritic cells, foam cells, mast cells, and neutrophilic granulocytes, and the number of 5-lipoxygenase expressing cells markedly increased in advanced lesions. By contrast, reticulocyte-type 15-lipoxygenase was expressed at levels that were several orders of magnitude lower than 5-lipoxygenase in both normal and diseased arteries, and its expression could not be related to lesion pathology. Our data support a model of atherogenesis in which 5-lipoxygenase cascade-dependent inflammatory circuits consisting of several leukocyte lineages and arterial wall cells evolve within the blood vessel wall during critical stages of lesion development. They raise the possibility that antileukotriene drugs may be an effective treatment regimen in late-stage disease.

Expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis.

Inflammatory infiltrates and atherosclerotic lesions emerge when monocytes adhere to endothelial cells (ECs), migrate into the subendothelial space, and become macrophages (MPhi(s)). Leukotrienes (LTs), products of 5-lipoxygenase, are powerful inflammatory mediators. 5-lipoxygenase+ MPhi(s) have been shown to increase during atherogenesis, and LT receptor (LT-R) transcripts were identified in diseased arteries. To investigate LT-Rs in cells involved in inflammation and atherogenesis, we used the in vitro models of human umbilical vein ECs (HUVECs) and monocyte-derived MPhi(s).

Differential leukotriene receptor expression and calcium responses in endothelial cells and macrophages indicate 5-lipoxygenase-dependent circuits of inflammation and atherogenesis.

Spreading depression (SD) is a slowly propagating wave of neuronal depolarization altering ion homeostasis, blood flow and energy metabolism without causing irreversible damage of the tissue. As SD has been implicated in several neurological diseases including migraine and stroke, understanding these disorders requires systematic knowledge of the processes modified by SD. Thus, we induced repetitive SD in the rat cerebral cortex by topical application of 3 m KCl for approximately 2 h and evaluated the kinetics of SD-induced changes in cortical gene expression for up to 30 days using Affymetrix RAE230A arrays. The temporal profile showed a rapid expression of immediate early genes, genes associated with inflammation, metabolism, stress and DNA repair, ion transport, and genes that play a role in growth/differentiation. Stress-response genes could still be detected after 24 h. At this time, induced genes were mainly related to the cell membrane and adhesion, or to the cytoskeleton. A subset of genes was still affected even 30 days after SD. Real-time polymerase chain reactions and immunohistochemistry confirmed the microarray results for several of the transcripts. Our findings demonstrate a temporal pattern of gene expression which might promote tissue remodeling and cortical plasticity, and might probably account for the mediation of neuronal tolerance towards subsequent ischemia.

Microarray-based long-term detection of genes differentially expressed after cortical spreading depression.

Spreading depression (SD) is an epiphenomenon of neurological disorders, like stroke or traumatic brain injury. These diseases have been associated with an increased neurogenesis in the adult rodent dentate gyrus. Such proliferative activity can also be induced by conditions that--like SD--coincide with a disturbed neuronal excitability, eg, epilepsy. Thus we hypothesized that SD might likewise influence hippocampal neurogenesis and potentially act as mediator of injury-induced neurogenesis.

Induction of neurogenesis in the adult dentate gyrus by cortical spreading depression.

GABA (gamma-aminobutyric acid), the main inhibitory neurotransmitter in the brain, is synthesized by glutamic acid decarboxylase (GAD). GAD exists in two adult isoforms, GAD65 and GAD67. During embryonic brain development at least two additional transcripts exist, I-80 and I-86, which are distinguished by insertions of 80 or 86 bp into GAD67 mRNA, respectively. Though it was described that embryonic GAD67 transcripts are not detectable during adulthood there are evidences suggesting re-expression under certain pathological conditions in the adult brain. In the present study we systematically analyzed for the first time the spatiotemporal distribution of different GADs with emphasis on embryonic GAD67 mRNAs in the postnatal brain using highly sensitive methods.

Adult and embryonic GAD transcripts are spatiotemporally regulated during postnatal development in the rat brain.

Adult neurogenesis continuously adds new neurons to the dentate gyrus and the olfactory bulb. It involves the proliferation and subsequent differentiation of neuronal progenitors, and is thus closely linked to the cell cycle machinery. Cell cycle progression is governed by the successive expression, activation and degradation of regulatory proteins. Among them, D-type cyclins control the exit from the G1 phase of the cell cycle. Cyclin D2 (cD2) has been shown to be required for the generation of new neurons in the neurogenic niches of the adult brain. It is differentially expressed during hippocampal development, and adult cD2 knock out (cD2KO) mice virtually lack neurogenesis in the dentate gyrus and olfactory bulb. In the present study we examined the dynamics of postnatal and adult neurogenesis in the dentate gyrus (DG) of cD2KO mice. Animals were injected with bromodeoxyuridine at seven time points during the first 10 months of life and brains were immunohistochemically analyzed for their potential to generate new neurons.

Age-dependent kinetics of dentate gyrus neurogenesis in the absence of cyclin D2.

There is a broad discussion concerning the function of new neurons in the adult brain. An increasingly accepted hypothesis proposes their crucial role in spatial learning. In this work, however, we demonstrate adult cyclin D2 knockout (cD2 KO) mice, which lack adult hippocampal neurogenesis, are able to learn a spatial version of the Barnes maze. Similar to wild type (WT) controls, these mutant mice exhibited several indicators of learning during 6 days of training: successively shorter latency and distance, higher speed, and decreasing number of errors. WT and cD2 KO mice showed improved search strategies, which became increasingly spatial. During probe Trial 1, mutant mice attained the highest significant number of nose-pokes at the former target hole compared with all the other holes. Both WT and cD2 KO mice covered shorter distances during probe Trial 2, whereas the mutant mice showed higher speed. We also discuss the possibility that some of the observed differences displayed by cD2 KO mice during training and at the probe trials-for example, longer mean distance and more errors-are associated with a smaller hippocampal formation. Our results suggest that adult brain neurogenesis is not obligatory for learning the Barnes maze.

Cyclin D2 knockout mice with depleted adult neurogenesis learn Barnes maze task.

The purpose of the present study was to determine whether a unilateral photothrombotic brain lesion induces bilateral ischemic tolerance towards a subsequent severe ischemia performed 5 days later. Severe ischemia was induced by transient (1h; t) or permanent (p) occlusion of the middle cerebral artery (MCAO). Rats were sacrificed 24h later. Preconditioning reduced the size of subsequent infarcts in both hemispheres. This effect was most prominent with tMCAO, and ipsilateral preconditioning was more effective than contralateral preconditioning (% of hemispheric volume, mean±SD: 31.9±3.7 to 19.0±10.3 with ipsilateral tMCAO; 31.9±3.7 to 22.9±4.9 with contralateral tMCAO; 64.7±4.3% to 47.2±12.5% with ipsilateral pMCAO; 64.7±4.3% to 53.1±8.9% with contralateral pMCAO). Ischemic preconditioning was associated with a successive bilateral up-regulation of superoxide dismutases which may be involved in the development of ischemic tolerance. Our data suggest that a focal ischemic brain lesion induces neuroprotective mechanisms in extensive brain areas and thus cause bilateral ischemic tolerance within a certain time window.

Bihemispheric ischemic tolerance induced by a unilateral focal cortical lesion.

Recently, we showed that cortical spreading depolarizations (CSDs) are a potent trigger of hippocampal neurogenesis. Here, we evaluated CSD-induced cytogenesis in the entorhinal cortex (EC), which provides the major afferent input to the dentate gyrus. Cortical spreading depolarizations were induced by epidural application of 3 mol/L KCl, controls received equimolar NaCl. Cytogenesis was analyzed at different time points thereafter by means of intraperitoneal 5-bromodeoxyuridine injections (day 2, 4, or days 1 to 7) and immunohistochemistry. Recurrent CSD significantly increased numbers of newborn cells in the ipsilateral EC. The majority of these cells expressed glial markers. Microglia proliferation was maximal at day 2, whereas NG2 glia and astrocytes responded for a prolonged period of time (days 2 to 4). Newborn glia remained detectable for 6 weeks after CSD. Whereas we furthermore detected newborn cells immunopositive for doublecortin, a marker for immature neuronal cells, we found no evidence for the generation of new neurons in the EC. Our results indicate that CSD is a potent gliogenic stimulus, leading to rapid and enduring changes in the glial cellular composition of the affected brain tissue. Thus, CSD facilitates ongoing structural remodeling of the directly affected cortex that might contribute to the pathophysiology of CSD-related brain pathologies.

Article	Year
Expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 12552108	2003
Differential leukotriene receptor expression and calcium responses in endothelial cells and macrophages indicate 5-lipoxygenase-dependent circuits of inflammation and atherogenesis. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 12816882	2003
Microarray-based long-term detection of genes differentially expressed after cortical spreading depression. The European journal of neuroscience\| PubMed ID: 16930413	2006
Induction of neurogenesis in the adult dentate gyrus by cortical spreading depression. Stroke; a journal of cerebral circulation\| PubMed ID: 18802207	2008
Adult and embryonic GAD transcripts are spatiotemporally regulated during postnatal development in the rat brain. PloS one\| PubMed ID: 19190758	2009
Age-dependent kinetics of dentate gyrus neurogenesis in the absence of cyclin D2. BMC neuroscience\| PubMed ID: 22564330	2012
Cyclin D2 knockout mice with depleted adult neurogenesis learn Barnes maze task. Behavioral neuroscience\| PubMed ID: 23244288	2013
Bihemispheric ischemic tolerance induced by a unilateral focal cortical lesion. Brain research\| PubMed ID: 24835408	2014
Cortical spreading depolarization stimulates gliogenesis in the rat entorhinal cortex. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism\| PubMed ID: 25515215	2015

Anja Urbach

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