Elimination of Microglia Improves Functional Outcomes Following Extensive Neuronal Loss in the Hippocampus

Abstract

With severe injury or disease, microglia become chronically activated and damage the local brain environment, likely contributing to cognitive decline. We previously discovered that microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for survival in the healthy adult brain, and we have exploited this dependence to determine whether such activated microglia contribute deleteriously to functional recovery following a neuronal lesion. Here, we induced a hippocampal lesion in mice for 25 d via neuronal expression of diphtheria toxin A-chain, producing both a neuroinflammatory reaction and behavioral alterations. Following the 25 d lesion, we administered PLX3397, a CSF1R inhibitor, for 30 d to eliminate microglia. This post-lesion treatment paradigm improved functional recovery on elevated plus maze and Morris water maze, concomitant with reductions in elevated proinflammatory molecules, as well as normalization of lesion-induced alterations in synaptophysin and PSD-95. Further exploration of the effects of microglia on synapses in a second cohort of mice revealed that dendritic spine densities are increased with long-term microglial elimination, providing evidence that microglia shape the synaptic landscape in the adult mouse brain. Furthermore, in these same animals, we determined that microglia play a protective role during lesioning, whereby neuronal loss was potentiated in the absence of these cells. Collectively, we demonstrate that microglia exert beneficial effects during a diphtheria toxin-induced neuronal lesion, but impede recovery following insult.

Significance statement: It remains unknown to what degree, and by what mechanisms, chronically activated microglia contribute to cognitive deficits associated with brain insults. We induced a genetic neuronal lesion in mice for 25 d and found activated microglia to increase inflammation, alter synaptic surrogates, and impede behavioral recovery. These lesion-associated deficits were ameliorated with subsequent microglial elimination, underscoring the importance of developing therapeutics aimed at eliminating/modulating chronic microglial activation. Additionally, we found long-term microglial depletion globally increases dendritic spines by ∼35% in the adult brain, indicating that microglia continue to sculpt the synaptic landscape in the postdevelopmental brain under homeostatic conditions. Microglial manipulation can therefore be used to investigate the utility of increasing dendritic spine numbers in postnatal conditions displaying synaptic aberrations.

Keywords: behavior; glia; inflammation; lesion; spines; synapse.

Figure 1.

Lesion-induced activation does not affect dependence on CSF1R signaling for survival in microglia: 5 to 8-month-old CaM/Tet mice (C57BL/6) underwent a 25 d neuronal lesion and subsequent microglial elimination via treatment with 290 mg/kg PLX3397. A, B, Schematics of CaM/Tet mouse model of inducible neuronal loss and experimental design. C, D, Thickness of the CA1 layer is significantly decreased with lesion (via two-way ANOVA, main effect of lesion p < 0.0001, interaction p = 0.0151). F, Number of microglia increases >200% with lesion, but subsequent PLX3397 treatment eliminates 85% of all cells. G, H, Representative 63× IBA1 immunofluorescent staining from the hippocampal region. I, J, 10× GFAP immunofluorescent staining of CA1 and dentate gyrus reveals increases in GFAP⁺ cells in lesion + PLX3397. Symbols denote significant differences between groups (p < 0.05): †control versus PLX3397; *control versus lesion; φPLX3397 versus lesion + PLX3397; #lesion versus lesion + PLX3397. Error bars indicate SEM; n = 7–10/group. S oriens, Stratum oriens; CA, cornus ammonis; S rad, stratum radiatum; S l-m, stratum lacunosum moleculare; L mol, molecular layer; DG, dentate gyrus.

Figure 2.

Neuronal lesioning does not induce peripheral cell migration into the brain: A, IL-1β levels significantly decrease with 70% microglial elimination in 2-month-old wild-type mice (n = 4/group). B–F, An additional cohort of 11 month-old CaM/Tet mice (B57BL/6) had doxycycline withdrawn from the diet for 25 d to induce a neuronal lesion in mice with both transgenes (lesion), and EB was injected on the 25^th day of lesion, 6 h before kill. Single-transgenic mice served as nonlesioning controls (control). B, EB staining was present in the peripheral tissue of all injected mice, but not in the brains of control or lesion mice. C, Quantification of EB absorbance at 595 nm. D, E, Representative flow cytometry analyses for PE-Cy7-CD11b and PE-CD45 in control and lesion mice. Box 1: live cells; Box 2: CD11b⁺ cells; Box 3: CD11b⁺, CD45⁺ cells; Box 4: CD45^lo/mod cells; Box 5: CD45^hi cells. F, Quantification revealed no differences between groups in the percentage of CD11b⁺ cells that were also CD45⁺ (Box 3), nor in the percentage of CD45^hi cells (Box 5). Mean PE intensity of CD45^lo/mod cells was significantly higher in lesion compared with control (two-tailed unpaired t test, p < 0.001; Box 4). G, Twenty times (top row) and 63× (bottom row) representative images show that regardless of experimental group, IBA1⁺ cells (red channel) in the brain did not stain positively for CD169 (green channel). CD169 positively labeled cells in thymus tissue. *p < 0.05, ****p < 0.001. Error bars indicate SEM; n = 3/group.

Figure 3.

Treatment with PLX3397 reverses lesion-induced increases in inflammatory signaling: Hippocampal mRNA levels for inflammatory transcripts were assessed by real-time PCR; dotted line indicates control, set to 100%. A, mRNA transcript levels of Il-15, Il-1α, and Socs1 were significantly increased with lesion compared with control animals. Il-15, Il-18, Il-1α, Il-6, and Socs1 were reduced in animals that were lesioned and treated with PLX3397, compared with animals that were just lesioned. Il-12α, Stat3, and Stat6 are significantly decreased with lesion compared with control animals. B, B2m, Bcl2, Bcl2l1, Ece1, Fn1, Ski, Smad3, and Vcam mRNA transcript levels were all significantly decreased with PLX3397 treatment, compared with untreated mice. B2m and Bax levels were increased with lesion and restored with PLX3397 treatment, whereas transcript levels of Cd34, Ece1, Ski, Smad3, Tfrc, and Vegf-α were all decreased with lesion alone compared with control animals. C, Ccl19 was increased with lesion, compared with control mice, and this effect was inhibited with PLX3397 treatment in lesioned mice. D, Col4a5, Nfkb2, and Ptgs2 levels were decreased with lesion, whereas Nfkb2 and Ptgs2 levels were also decreased with microglial elimination. E, Levels of Ccr2, Cd80, Cd86, H2eb1, and Ptprc were all increased with lesion, compared with control mice, whereas PLX3397 treatment restored these effects in lesioned mice. Cd4, Cd86, and H2eb1 were all significantly decreased with PLX3397 treatment alone. F, Cd34 was significantly reduced with both PLX3397 treatment and lesion compared with control mice. Symbols denote significant differences between groups (p < 0.05): †control versus PLX3397; *control versus lesion; φPLX3397 versus lesion + PLX3397; #lesion versus lesion + PLX3397. Error bars indicate SEM; n = 4/group.

Figure 4.

Neuronal lesioning induces synaptic alterations that are restored with subsequent microglial elimination: A, Representative 63× images of PSD95 and synaptophysin immunoreactivity in the CA1 region display alterations in puncta area and size. B–D, Quantification of PSD95 puncta number, area, and intensity in the CA1 shows that microglial elimination restores most lesion-induced alterations. E–G, Quantification of synaptophysin puncta number, area, and intensity in the CA1 shows lesion-induced alterations are reversed with PLX3397 treatment. H, Representative 63× images of PSD95 and synaptophysin immunoreactivity in the stratum radiatum. I–K, Quantification of PSD95 puncta number, area, and intensity in the stratum radiatum reveals increases in puncta number with microglial elimination. L–N, Quantification of synaptophysin puncta number, area, and intensity in stratum radiatum reveals changes in puncta area and intensity with PLX3397 and/or lesion. Symbols denote significant differences between groups (p < 0.05): †control versus PLX3397; *control versus lesion; φPLX3397 versus lesion + PLX3397; #lesion versus lesion + PLX3397. Error bars indicate SEM; n = 7–10/group.

Figure 5.

Elimination of microglia following extensive neuronal loss improves functional recovery: A, Lesion mice spent less time in closed arms and more time in open arms on the elevated plus maze test, compared with control mice (via two-way ANOVA, main effect of lesion p = 0.0006). Lesion + PLX3397 mice performed similarly to control and PLX3397 groups in both closed and exposed arms. B, All four groups of mice had comparable numbers of arm entries. C, All four groups of mice spent similar amounts of time in the middle arena of open field testing apparatus. D, All mice learned over the 5 d training period, though the lesion group trended to a longer latency to find platform on Day 5, compared with lesion + PLX3397 group (p = 0.0882). E, Treatment with PLX3397 increased performance on the probe trial (via two-way ANOVA, main effect of treatment p = 0.0007). F, G, All four groups of mice demonstrated comparable swim speeds and distance traveled during the probe trial. Symbols denote significant differences between groups (p < 0.05): †control versus PLX3397; *control versus lesion; φPLX3397 versus lesion + PLX3397; #lesion versus lesion + PLX3397. Error bars indicate SEM; n = 7–10/group.

Figure 6.

Microglial elimination in thy1-GFP-M expressing CaM/Tet mice: 5- to 8-month-old GFP-CaM/Tet mice (C57BL/6-CBA mix) were treated with 290 mg/kg PLX3397 for 2 months to deplete microglia. A 25 d neuronal lesion was induced in one-half of the animals at the same time treatment with PLX3397 began. A, B, Schematics of GFP-CaM/Tet mouse model of inducible neuronal loss and experimental design. C, Representative 10× confocal images of the hippocampal region from each group for neuronal nuclei (NEUN in blue channel), microglia (IBA1 in red channel), and GFP in the green channel. D, Representative 63× IBA1 immunofluorescent staining from the hippocampal region showing rod like morphologies with lesion. E, Quantification of microglia in the field-of-view by automated, direct cell counting reveals a significant decrease in both PLX3397-treated groups. F, Estimation of microglia per hippocampus by stereological methods also reveals a significant decrease in both PLX3397-teated groups. G, Quantification of the thickness of the hippocampal layers reveals a significant lesion-induced decrease only in the stratum radiatum. H, Representative 20× pictures of Cresyl Violet staining in the CA1 cell layer from each group. I, Stereological estimation of neurons in the CA1 region from each group reveals a significant decrease only in the lesion + PLX3397 group. J, Lesion mice did not display a deficit in performance on elevated plus maze. Symbols denote significant differences between groups (p < 0.05): †control versus PLX3397; *control versus lesion; φPLX3397 versus lesion + PLX3397; #lesion versus lesion + PLX3397. Error bars indicate SEM; n = 5–6/group.

Figure 7.

Microglial elimination increases dendritic spine densities throughout the brain. A, Representative images of dendrites from the CA1 region. B, Quantification of dendritic spines, overall and by type, in the CA1 region reveals lesion- and PLX3397-associated increases. C, Representative images of dendrites from layer 5 in V1. D, Quantification of dendritic spines, overall and by type, in layer 5 of V1 reveals only PLX3397-associated increases. Symbols denote significant differences between groups (p < 0.05): †control versus PLX3397; *control versus lesion; φPLX3397 versus lesion + PLX3397; #lesion versus lesion + PLX3397. Error bars indicate SEM; n = 5/group.