Spared nerve injury後のマウス後根神経節におけるNGFとBDNFの発現

Neuropathic pain is initiated by a primary lesion in the peripheral nervous system and spoils quality of life. Neurotrophins play important roles in the development and transmission of neuropathic pain. There are conflicting reports that the dorsal root ganglion (DRG) in an injured nerve contribute to neuropathic pain, whereas several studies have highlighted the important contribution of the DRG in a non-injured nerve. Clarifying the role of neurotrophins in neuropathic pain is problematic because we cannot distinguish injured and intact neurons in most peripheral nerve injury models. In the present study, to elicit neuropathic pain, we used the spared nerve injury (SNI) model, in which injured DRG neurons are distinguishable from intact ones, and mechanical allodynia develops in the intact sural nerve skin territory. We examined nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression in the DRGs of SNI model mice. NGF and BDNF levels increased in the injured L3 DRG, while NGF decreased in the intact L5 DRG. These data offer a new point of view on the role of these neurotrophins in neuropathic pain induced by peripheral nerve injury.博士（医学）・甲第698号・平成31年3月15日© 2018 Elsevier B.V. All rights reserved

Olig2-Lineage Astrocytes: A Distinct Subtype of Astrocytes That Differs from GFAP Astrocytes

Astrocytes are the most abundant glia cell type in the central nervous system (CNS), and are known to constitute heterogeneous populations that differ in their morphology, gene expression and function. Although glial fibrillary acidic protein (GFAP) is the cardinal cytological marker of CNS astrocytes, GFAP-negative astrocytes can easily be found in the adult CNS. Astrocytes are also allocated to spatially distinct regional domains during development. This regional heterogeneity suggests that they help to coordinate post-natal neural circuit formation and thereby to regulate eventual neuronal activity. Here, during lineage-tracing studies of cells expressing Olig2 using Olig2CreER; Rosa-CAG-LSL-eNpHR3.0-EYFP transgenic mice, we found Olig2-lineage mature astrocytes in the adult forebrain. Long-term administration of tamoxifen resulted in sufficient recombinant induction, and Olig2-lineage cells were found to be preferentially clustered in some adult brain nuclei. We then made distribution map of Olig2-lineage astrocytes in the adult mouse brain, and further compared the map with the distribution of GFAP-positive astrocytes visualized in GFAPCre; Rosa-CAG-LSL-eNpHR3.0-EYFP mice. Brain regions rich in Olig2-lineage astrocytes (e.g., basal forebrain, thalamic nuclei, and deep cerebellar nuclei) tended to lack GFAP-positive astrocytes, and vice versa. Even within a single brain nucleus, Olig2-lineage astrocytes and GFAP astrocytes frequently occupied mutually exclusive territories. These findings strongly suggest that there is a subpopulation of astrocytes (Olig2-lineage astrocytes) in the adult brain, and that it differs from GFAP-positive astrocytes in its distribution pattern and perhaps also in its function. Interestingly, the brain nuclei rich in Olig2-lineage astrocytes strongly expressed GABA-transporter 3 in astrocytes and vesicular GABA transporter in neurons, suggesting that Olig2-lineage astrocytes are involved in inhibitory neuronal transmission

Propofol induces nuclear localization of Nrf2 under conditions of oxidative stress in cardiac H9c2 cells

<div><p>Oxidative stress contributes to myocardial ischemia-reperfusion injury, which causes cardiomyocyte death and precipitate life-threatening heart failure. Propofol has been proposed to protect cells or tissues against oxidative stress. However, the mechanisms underlying its beneficial effects are not fully elucidated. In the present study, we employed an <i>in vitro</i> oxidative injury model, in which rat cardiac H9c2 cells were treated with H₂O₂, and investigated roles of propofol against oxidative stress. Propofol treatment reduced H₂O₂-induced apoptotic cell death. While H₂O₂ induced expression of the antioxidant enzyme HO-1, propofol further increased HO-1 mRNA and protein levels. Propofol also promoted nuclear localization of Nrf2 in the presence of H₂O₂. Knockdown of Nrf2 using siRNA suppressed propofol-inducible Nrf2 and expression of Nrf2-downstream antioxidant enzyme. Knockdown of Nrf2 suppressed the propofol-induced cytoprotection. In addition, Nrf2 overexpression induced nuclear localization of Nrf2 and HO-1 expression. These results suggest that propofol exerts antioxidative effects by inducing nuclear localization of Nrf2 and expression of its downstream enzyme in cardiac cells. Finally, we examined the effect of propofol on cardiomyocytes using myocardial ischemia-reperfusion injury models. The expression level of Nrf2 protein was increased at 15 min after reperfusion in the ischemia-reperfusion and propofol group compared with ischemia-reperfusion group in penumbra region. These results suggest that propofol protects cells or tissues from oxidative stress via Nrf2/HO-1 cascade.</p></div

Propofol upregulate HO-1 expression and nuclear localization of Nrf2 under oxidative stress conditions.

<p><b>A</b>: mRNA expression profile for HO-1. n = 3. The data are expressed as mean ± SEM. <b>B</b>: Western blot analysis of HO-1 in H9c2 cells. <b>C</b>: Quantification of the density of expression level of HO-1. n = 3. The data are expressed as mean ± SEM. <b>D</b>: Western blot analysis of Nrf2 in H9c2 cells. <b>E</b>: Quantification of the density of expression level of total Nrf2. n = 3. The data are expressed as mean ± SEM. <b>F</b>: Western blot analysis of Nrf2 in total lysates, nuclear lysates and cytoplasmic lysates of H9c2 cells. <b>G</b>: Western blot analysis of Nrf2 in nuclear lysates and cytoplasmic lysates of H9c2 cells. <b>H</b>, <b>I</b>: Quantification of the density of expression level of nuclear Nrf2 and cytoplasmic Nrf2. <b>J</b>: Representative immunocytochemical labeling of Nrf2 in H9c2 cells treated with H₂O₂ and propofol (PR). Scale bar, 50 μm. Control (con) means no treatment. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Effects of propofol on myocardial ischemia-reperfusion injury.

<p><b>A-B</b>: Scheme of ischemia-reperfusion injury and propofol administration. <b>C</b>: Expression profiles of HO-1 mRNA in penumbra and infarction region of ischemia-reperfusion (I/R) group rats and I/R+propofol group rats at 15 min or 90 min after reperfusion. Rep, reperfusion. n = 4. The data are expressed as mean ± SEM. <b>D</b>: Western blot analysis of Nrf2 in penumbra and infarction region of I/R group rats and I/R+propofol group rats at 15 min after reperfusion. n = 3. The data are expressed as mean ± SEM.</p

Nrf2 overexpression induce nuclear localization of Nrf2 and HO-1 expression.

<p><b>A</b>: Western blot analysis of Nrf2 in HEK293T cells using anti-c-Myc antibody. <b>B</b>: Western blot analysis of Nrf2 in H9c2 cells. Upper band is nuclear Nrf2 (N-Nrf2) and lower band is cytoplasmic Nrf2 (C-Nrf2). <b>C</b>: Quantification of the density of expression level of nuclear Nrf2. n = 3. The data are expressed as mean ± SEM. <b>D</b>: Western blot analysis of Nrf2 and HO-1 in Nrf2 overexpressed H9c2 cells. The number means intensity of nuclear Nrf2 (arrows). <b>E</b>: Quantification of the density of expression level of nuclear Nrf2. n = 3. The data are expressed as mean ± SEM. *<i>P</i> < 0.05.</p

Propofol inhibits H₂O₂-induced H9c2 cell death.

<p><b>A</b>: Effect of H₂O₂ on cell viability. Cytotoxicity was measured using an LDH assay. n = 3. <b>B</b>: Effect of propofol (PR) on cell viability. Scale bar, 200 μm. <b>C</b>: Quantitative analysis of PR-induced cytotoxicity in H9c2 cells. n = 3. <b>D</b>: Diagram of the scheme of propofol (PR) and H₂O₂ treatment. <b>E</b>: Effect of PR on cell viability under oxidative stress conditions. Arrows, dead cells; arrowheads, surviving cells. Scale bar, 250 μm. <b>F</b>: Quantification of dead H9c2 cells. n = 3. The data are expressed as mean ± SEM. <b>G</b>: TUNEL staining. Arrowheads, TUNEL-positive cells. Scale bar, 200 μm. <b>H</b>: Quantification of TUNEL-positive cells. n = 3. The data are expressed as mean ± SEM. Control (con) means no treatment. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Nrf2 inhibition suppresses propofol-induced cytoprotection.

<p><b>A-D</b>: Effect of Nrf2, HO-1, Nqo-1 or scramble siRNA on propofol-induced cytoprotection, after at 24 h after treatment with H₂O₂. Cytotoxicity was measured using an LDH assay. n = 3. The data are expressed as mean ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01. N.S., not significant.</p

Knockdown of Nrf2 by siRNA suppresses propofol-induced antioxidant expression.

<p><b>A</b>: Expression of Nrf2 mRNA in H9c2 cells transfected with Nrf2 and scramble (control) siRNA. n = 3. The data are expressed as mean ± SEM. <b>B-D</b>: Expression profiles of Nrf2 (B), HO-1 (C) and Nqo-1 (D) mRNAs in H9c2 cells transfected with scramble, Nrf2 and HO-1 siRNA. n = 3. The data are expressed as mean ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p