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

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

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

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

Temporal profile of cell proliferation in the SGZ of DG.

<p>Quantification of BrdU⁺ cells in the SGZ of the DG at 3, 6 and 24 hours after the end of OGD. Each column shows the total number of BrdU⁺ cells in the SGZ. Bars represent the mean±SEM. In parentheses is the number of slices investigated. *<i>P</i><0.05 and **<i>P</i><0.01 <i>vs</i> control, One-way ANOVA followed by Newman–Keuls post hoc test.</p

The Selective Antagonism of P2X₇ and P2Y₁ Receptors Prevents Synaptic Failure and Affects Cell Proliferation Induced by Oxygen and Glucose Deprivation in Rat Dentate Gyrus

<div><p>Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X₇ and P2Y₁ receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y₁ receptor) and BBG (selective antagonist of P2X₇ receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2′-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU⁺ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU⁺ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y₁ antagonist reduced the number of BrdU⁺ cells at this time. The data demonstrate that P2X₇ and P2Y₁ activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y₁ receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.</p></div

Quantitative analysis of neuronal debris in Str. Radiatum, involvement of Cx43 in astrocytes-neuron interplay.

<p>Images from CA1 Str. Pyramidalis and CA1 Str. Radiatum of an adult (<b>A</b>), aged (<b>B</b>) and LPS-treated rat (<b>C</b>) showing the presence of neuronal debris (arrows, <b>B</b> and <b>C</b>). Scale bar: 70 µm. <b>D1–D3:</b> higher magnification images of GFAP (green, <b>D1</b>) and NeuN (red, <b>D2</b>) staining and the merge of the two previous images (<b>D3</b>). Empty arrows show neuronal debris closely apposed to astrocyte branches. Scale bar: 15 µm<b>. E:</b> quantitative analysis of neuronal debris in CA1 Str. Radiatum of adult (n = 12), aged (n = 10), aCSF- (n = 5) and LPS-treated (n = 6) rats (mean±SEM; *** and ^###P<0.001 vs all other groups). <b>F–F4</b>: Representative images of triple immunostaining of GFAP (green), NeuN (red) and Cx43 (blue) in the Str. Radiatum of an aged rat. <b>F</b>: 3D stack of 39 confocal scans (total 14.39 µm); <b>F1</b>: a “sub-slice” of the previous neuron (obtained stacking 6 consecutive scans, total 1.843 µm, starting at a depth of 5.899 µm into the cell) and separate staining of Cx43 (<b>F2</b>), GFAP (<b>F3</b>) and NeuN (<b>F4</b>). Scale bar: 5 µm (<b>F)</b>; 10 µm (<b>F1–F4).</b></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

Quantitative analysis of astrocytes in CA1 Str. Radiatum.

<p>Characterization of astrocytes in CA1 Str. Radiatum of adult and aged rats. Representative epifluorescent photomicrographs showing immunoreactivity of GFAP (green) and DAPI staining (blue) in CA1 Pyramidal cell layer and Str. Radiatum of adult (<b>A1,A2,A3</b>) and aged (<b>B1,B2,B3</b>) rats. <b>A3</b> and <b>B3</b> show the merged images. Scale bar: 50 µm. <b>C:</b> quantitative analysis of GFAP positive cells counted in CA1 Str. Radiatum of adult (n = 12), aged (n = 15), aCSF- (n = 5) and LPS-treated (n = 6) rats, expressed as GFAP positive cells/mm² (mean±SEM); **P<0.01 vs all other groups. <b>D:</b> length of principal astrocyte branches in CA1 Str. Radiatum of adult (n = 12), aged (n = 15), aCSF- (n = 5) and LPS-treated (n = 6) rats; (mean±SEM), **P<0.01 vs all other groups.</p