3 research outputs found

    Image2.TIF

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    <p>Previously, we have shown that the transcription factor nuclear factor interleukin (NF-IL)6 can be used as an activation marker for inflammatory lipopolysaccharide (LPS)-induced and psychological novel environment stress (NES) in the rat brain. Here, we aimed to investigate age dependent changes of hypothalamic and pituitary responses to NES (cage switch) or LPS (100 μg/kg) in 2 and 24 months old rats. Animals were sacrificed at specific time points, blood and brains withdrawn and analyzed using immunohistochemistry, RT-PCR and bioassays. In the old rats, telemetric recording revealed that NES-induced hyperthermia was enhanced and prolonged compared to the young group. Plasma IL-6 levels remained unchanged and hypothalamic IL-6 mRNA expression was increased in the old rats. Interestingly, this response was accompanied by a significant upregulation of corticotropin-releasing hormone mRNA expression only in young rats after NES and overall higher plasma corticosterone levels in all aged animals. Immunohistochemical analysis revealed a significant upregulation of NF-IL6-positive cells in the pituitary after NES or LPS-injection. In another important brain structure implicated in immune-to-brain communication, namely, in the median eminence (ME), NF-IL6-immunoreactivity was increased in aged animals, while the young group showed just minor activation after LPS-stimulation. Interestingly, we found a higher amount of NF-IL6-CD68-positive cells in the posterior pituitary of old rats compared to the young counterparts. Moreover, aging affected the regulation of cytokine interaction in the anterior pituitary lobe. LPS-treatment significantly enhanced the secretion of the cytokines IL-6 and TNFα into supernatants of primary cell cultures of the anterior pituitary. Furthermore, in the young rats, incubation with IL-6 and IL-10 antibodies before LPS-stimulation led to a robust decrease of IL-6 production and an increase of TNFα production by the pituitary cells. In the old rats, this specific cytokine interaction could not be detected. Overall, the present results revealed strong differences in the activation patterns and pathways between old and young rats after both stressors. The prolonged hyperthermic and inflammatory response seen in aged animals seems to be linked to dysregulated pituitary cytokine interactions and brain cell activation (NF-IL6) in the hypothalamus-pituitary-adrenal axis.</p

    Image1.TIF

    No full text
    <p>Previously, we have shown that the transcription factor nuclear factor interleukin (NF-IL)6 can be used as an activation marker for inflammatory lipopolysaccharide (LPS)-induced and psychological novel environment stress (NES) in the rat brain. Here, we aimed to investigate age dependent changes of hypothalamic and pituitary responses to NES (cage switch) or LPS (100 μg/kg) in 2 and 24 months old rats. Animals were sacrificed at specific time points, blood and brains withdrawn and analyzed using immunohistochemistry, RT-PCR and bioassays. In the old rats, telemetric recording revealed that NES-induced hyperthermia was enhanced and prolonged compared to the young group. Plasma IL-6 levels remained unchanged and hypothalamic IL-6 mRNA expression was increased in the old rats. Interestingly, this response was accompanied by a significant upregulation of corticotropin-releasing hormone mRNA expression only in young rats after NES and overall higher plasma corticosterone levels in all aged animals. Immunohistochemical analysis revealed a significant upregulation of NF-IL6-positive cells in the pituitary after NES or LPS-injection. In another important brain structure implicated in immune-to-brain communication, namely, in the median eminence (ME), NF-IL6-immunoreactivity was increased in aged animals, while the young group showed just minor activation after LPS-stimulation. Interestingly, we found a higher amount of NF-IL6-CD68-positive cells in the posterior pituitary of old rats compared to the young counterparts. Moreover, aging affected the regulation of cytokine interaction in the anterior pituitary lobe. LPS-treatment significantly enhanced the secretion of the cytokines IL-6 and TNFα into supernatants of primary cell cultures of the anterior pituitary. Furthermore, in the young rats, incubation with IL-6 and IL-10 antibodies before LPS-stimulation led to a robust decrease of IL-6 production and an increase of TNFα production by the pituitary cells. In the old rats, this specific cytokine interaction could not be detected. Overall, the present results revealed strong differences in the activation patterns and pathways between old and young rats after both stressors. The prolonged hyperthermic and inflammatory response seen in aged animals seems to be linked to dysregulated pituitary cytokine interactions and brain cell activation (NF-IL6) in the hypothalamus-pituitary-adrenal axis.</p

    Table_1_Tracking cell turnover in human brain using 15N-thymidine imaging mass spectrometry.docx

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    Microcephaly is often caused by an impairment of the generation of neurons in the brain, a process referred to as neurogenesis. While most neurogenesis in mammals occurs during brain development, it thought to continue to take place through adulthood in selected regions of the mammalian brain, notably the hippocampus. However, the generality of neurogenesis in the adult brain has been controversial. While studies in mice and rats have provided compelling evidence for neurogenesis occurring in the adult rodent hippocampus, the lack of applicability in humans of key methods to demonstrate neurogenesis has led to an intense debate about the existence and, in particular, the magnitude of neurogenesis in the adult human brain. Here, we demonstrate the applicability of a powerful method to address this debate, that is, the in vivo labeling of adult human patients with 15N-thymidine, a non-hazardous form of thymidine, an approach without any clinical harm or ethical concerns. 15N-thymidine incorporation into newly synthesized DNA of specific cells was quantified at the single-cell level with subcellular resolution by Multiple-isotype imaging mass spectrometry (MIMS) of brain tissue resected for medical reasons. Two adult human patients, a glioblastoma patient and a patient with drug-refractory right temporal lobe epilepsy, were infused for 24 h with 15N-thymidine. Detection of 15N-positive leukocyte nuclei in blood samples from these patients confirmed previous findings by others and demonstrated the appropriateness of this approach to search for the generation of new cells in the adult human brain. 15N-positive neural cells were easily identified in the glioblastoma tissue sample, and the range of the 15N signal suggested that cells that underwent S-phase fully or partially during the 24 h in vivo labeling period, as well as cells generated therefrom, were detected. In contrast, within the hippocampus tissue resected from the epilepsy patient, none of the 2,000 dentate gyrus neurons analyzed was positive for 15N-thymidine uptake, consistent with the notion that the rate of neurogenesis in the adult human hippocampus is rather low. Of note, the likelihood of detecting neurogenesis was reduced because of (i) the low number of cells analyzed, (ii) the fact that hippocampal tissue was explored that may have had reduced neurogenesis due to epilepsy, and (iii) the labeling period of 24 h which may have been too short to capture quiescent neural stem cells. Yet, overall, our approach to enrich NeuN-labeled neuronal nuclei by FACS prior to MIMS analysis provides a promising strategy to quantify even low rates of neurogenesis in the adult human hippocampus after in vivo15N-thymidine infusion. From a general point of view and regarding future perspectives, the in vivo labeling of humans with 15N-thymidine followed by MIMS analysis of brain tissue constitutes a novel approach to study mitotically active cells and their progeny in the brain, and thus allows a broad spectrum of studies of brain physiology and pathology, including microcephaly.</p
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