Characterization of astrocytes-neurons interplay.

<p>Photos show confocal images of immunoreactivity of GFAP (green) and NeuN (red) in CA1 Pyramidal cell layer and CA1 Str. Radiatum of adult (<b>A</b>), aged (<b>B</b> and <b>D, E1–F3</b>) and LPS-treated rats (<b>C</b>). Scale bar: 60 µm (<b>A</b>,<b>B</b>,<b>C</b>). <b>D:</b> 3D stack of confocal scans of GFAP (green), and NeuN (red). Scale bar: 5 µm. <b>E1–E3:</b> each panel is obtained merging 2 consecutive confocal scans (total 0.738 µm). Scale bar: 5 µm. <b>F1–F3∶</b>3D stacks of the neuron shown in D, digitally cut along the white dotted line and rotated by 0, 45 and 90 degrees along the vertical axis. Scale bar: 3 µm.</p

Analysis of CX3CL1 expression in the hippocampus of adult, aged, aCSF- and LPS-treated rats.

<p><b>A1</b>: Western Blot analysis of CX3CL1 in whole hippocampus homogenates of adult (n = 7), aged (n = 4), aCSF- (n = 4) and LPS-treated (n = 4) rats. Each column in the graph represents the level of CX3CL1 expressed as mean±SEM, normalized to β-actin run in the same gel. *P<0.05 vs adult; #P<0.05 vs aCSF-tretated. <b>A2:</b> representative Western Blot runs of CX3CL1 and of β-actin. <b>B1–B3:</b> laser confocal microscopy immunohistochemistry of neurons (<b>B1,</b> NeuN, red), CX3CL1 (<b>B2,</b> green) and the merge of the two previous images (<b>B3</b>) from the CA1 region of an aged rat. Scale bar: 14 µm. <b>C1–C4:</b> epifluorescent microscopy images of a microglia cell (<b>C1</b>, red), CX3CL1 immunostaining (<b>C2,</b> green), DAPI staining of nuclei (<b>C3,</b> blue) and the merge of the three previous images (<b>C4</b>), indicating that CX3CL1 staining is localized on the surface of a cell, possibly a neuron (arrows). <b>D:</b> the image represents a confocal “sub-slice” (total thickness 2.233 µm) of the same microglia cell shown in C1–C4, acquired starting at a depth of 8.932 µm into the cell. The CX3CL1 positive cell (green) is partially colocalized with the microglia cell. Scale bar: 14 µm.</p

RLX and Notch-1 negatively regulates TGF-β1-induced fibroblast-myofibroblast transition in cardiac fibroblasts.

<p>Neonatal cardiac fibroblasts were cultured for 48 h and treated as indicated. A) Western blotting analysis of NICD expression in the absence (control) or presence of DAPT (5 µM) a pharmacological γ-secretase inhibitor, used to block the generation of NICD. B) Western Blotting analysis of α–sma and MMP-2 expression in the cells treated with DAPT. C) Representative confocal immunofluorescence images cardiac fibroblasts treated with DAPT, fixed and stained with antibodies against α–sma (green). Nuclei are marked in red with propidium iodide. D) Western blotting analysis of Jagged-1 expression in control cells, cells transfected with non specific scrambled-siRNA (SCR-siRNA) or silenced for the expression of Notch-1 ligand, Jagged-1, by specific Jagged-1 siRNA (Jagged-1 siRNA). E) Western Blotting analysis of α–sma in Jagged-1 silenced cells. F) Representative confocal immunofluorescence images cardiac fibroblasts silenced for Jagged-1 expression, fixed and stained with antibodies against α–sma (green). Nuclei are marked in red with propidium iodide. The densitometric analyses of the bands normalized to GAPDH are reported in histograms in A–E; the densitometric analyses α–sma fluorescent signal are shown in the histograms in and C, F. Significance of differences: *p<0.05 <i>vs</i> control, ^δp<0.05 vs SCR-siRNA, °p<0.05 <i>vs</i> TGF-β1, ^#p<0.05 vs TGF-β1+ RLX.</p

The Neuron-Astrocyte-Microglia Triad in Normal Brain Ageing and in a Model of Neuroinflammation in the Rat Hippocampus

<div><p>Ageing is accompanied by a decline in cognitive functions; along with a variety of neurobiological changes. The association between inflammation and ageing is based on complex molecular and cellular changes that we are only just beginning to understand. The hippocampus is one of the structures more closely related to electrophysiological, structural and morphological changes during ageing. In the present study we examined the effect of normal ageing and LPS-induced inflammation on astroglia-neuron interaction in the rat hippocampus of adult, normal aged and LPS-treated adult rats. Astrocytes were smaller, with thicker and shorter branches and less numerous in CA1 Str. radiatum of aged rats in comparison to adult and LPS-treated rats. Astrocyte branches infiltrated apoptotic neurons of aged and LPS-treated rats. Cellular debris, which were more numerous in CA1 of aged and LPS-treated rats, could be found apposed to astrocytes processes and were phagocytated by reactive microglia. Reactive microglia were present in the CA1 Str. Radiatum, often in association with apoptotic cells. Significant differences were found in the fraction of reactive microglia which was 40% of total in adult, 33% in aged and 50% in LPS-treated rats. Fractalkine (CX3CL1) increased significantly in hippocampus homogenates of aged and LPS-treated rats. The number of CA1 neurons decreased in aged rats. In the hippocampus of aged and LPS-treated rats astrocytes and microglia may help clearing apoptotic cellular debris possibly through CX3CL1 signalling. Our results indicate that astrocytes and microglia in the hippocampus of aged and LPS-infused rats possibly participate in the clearance of cellular debris associated with programmed cell death. The actions of astrocytes may represent either protective mechanisms to control inflammatory processes and the spread of further cellular damage to neighboring tissue, or they may contribute to neuronal damage in pathological conditions.</p> </div

Relaxin prevents TGF-β1 induced down regulation of MMP-2 and MMP-9 expression and up-regulation of TIMP-2 in NIH/3T3 and primary neonatal cardiac fibroblasts.

<p>A–C, F–H). Representative confocal immunofluorescence images of NIH/3T3 cells (A–C) and primary neonatal cardiac fibroblasts (F–H) cultured in the indicated experimental conditions and immunostained with antibodies against MMP-2 (A,F; cyan), MMP-9 (B,G; green) or TIMP-2 (C,H, green). In B and G the nuclei are labeled in red with propidium iodide. The histograms show the corresponding densitometric analyses of the intensity of MMP-2, MMP-9 and TIMP-2 fluorescence signals. D–E) Western blotting analyses of the expression of (D) MMP-2, and (E) MMP-9 proteins in neonatal cardiac fibroblasts. In the histograms the densitometric analyses of the bands normalized to GAPDH are reported. Data are representative of at least three independent experiments with similar results. Significance of differences: *p<0.05 <i>vs</i> control, °p<0.05 <i>vs</i> TGF-β1.</p

Relaxin attenuates TGF-β1 induced cytoskeletal assembly in NIH/3T3 and primary neonatal cardiac fibroblasts.

<p>Representative confocal immunofluorescence images of (A–D) NIH/3T3 cells stained with TRITC-phalloidin to reveal F-actin and of (E–H) primary cardiac fibroblasts stained with TRITC-phalloidin (red) and anti-vinculin antibody (green) to detect focal adhesions, cultured for 48 h in the indicated experimental conditions. I, J) Densitometric analyses of the intensity of F-actin and vinculin fluorescence signals performed on digitized images of neonatal cardiac fibroblasts. The images are representative of at least three independent experiments with similar results. Significance of differences: *p<0.05 <i>vs</i> control, °p<0.05 <i>vs</i> TGF-β.</p

Summary of results.

<p>The first data column reports the data obtained from the quantitative analyses performed in adult rat hippocampus, taken as controls: GFAP levels in hippocampal homogenates (ratio of ß-actin); GFAP positive cells in CA1 Str. Radiatum (number); Astrocyte branches in CA1 Str. Radiatum (µm); Debris in CA1 Str. Radiatum (number); Phospho-p38MAPK positive cells in CA1 Str. Pyramidalis (number); Total microglia in CA1 Str. Radiatum (number); Reactive microglia in CA1 Str. Radiatum (number); Resting microglia in CA1 Str. Radiatum (number); CX3CL1 levels in hippocampal homogenates (ratio of ß-actin); DAPI in CA1 Str. Pyramidalis (number); CA1 Str. Pyramidalis thickness (µm). All other data are reported as percent variations of those found in adult rats.</p

Immunostaining of markers of apoptosis in cells surrounded by astrocyte branches in CA1 Str. Radiatum.

<p><b>A1,A2:</b> immunostaining for CytC (red) and GFAP (green). <b>B1,B2:</b> immunostaining for AIF (red) and GFAP (green). µm. Representative images of immunostaining for NeuN (red), AIF (blue) and GFAP (green) taken from the CA1 region of an adult (<b>C1–C2</b>), an aged (<b>D1–D2</b>) and an LPS-treated (<b>E1–E2</b>) rat. Note the presence of AIF staining within neurons of aged and LPS treated rats only (open arrows in <b>D1,D2</b> and <b>E1,E2</b>). This effect was observed in all slices from aged and LPS-treated rats. Scale bar: 10. <b>F:</b> Quantification of phospho-p38MAPK positive cells in CA1 Str. Pyramidalis of adult (n = 11), aged (n = 16), aCSF- (n = 5) and LPS-treated (n = 5) rats (mean±SEM; ***P<0.001, vs all other groups).</p

Western Blot analysis of GFAP levels in hippocampus and immunohistochemistry of GFAP positive cells. A1:

<p>quantification of GFAP by Western Blot from homogenates of whole hippocampus. Each column represents the levels of GFAP expressed as a ratio of β-actin expression run in the same gel (mean ± SEM; Adult, n = 8; aged, n = 5; aCSF, n = 6; LPS-treated, n = 6) <b>A2:</b> representative Western Blot runs of GFAP and β-actin. <b>B,C</b>: immunolabelling of astrocytes using anti GFAP antibody and DAB staining in whole hippocampal slices. <b>B1–B3</b>: higher magnification images of CA1 (<b>B1</b>), CA3 (<b>B2</b>) and DG (<b>B3</b>); <b>C1–C3</b>: higher magnification images of CA1 (<b>C1</b>), CA3 (<b>C2</b>) and DG (<b>C3</b>); <b>B-B3</b>: adult rat; <b>C–C3</b>: aged rat. Scale bar: <b>B–C</b>: 400 µm; <b>B1–C3</b>∶70 µm.</p

Characterization and expression of RLX receptor on primary neonatal cardiac fibroblasts.

<p>A) Representative contrast phase microscopy images of first passage neonatal murine neonatal cardiac fibroblasts. B) Representative superimposed differential interference contrast (DIC) and confocal immunofluorescence images of neonatal cardiac fibroblasts immunostained with antibodies against vimentin (green). Nuclei are counterstained in red with propidium iodide. C) Expression of Relaxin family peptide receptor 1 (RXFP1) in neonatal cardiac fibroblasts and NIH/3T3 at mRNA level determined by RT-PCR, and protein level evaluated by Western blotting analysis. The images are representative of at least three independent experiments with similar results.</p