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. 2015 Aug 7;6(9):3303-12.
doi: 10.1364/BOE.6.003303. eCollection 2015 Sep 1.

In Vivo Imaging of Activated Microglia in a Mouse Model of Focal Cerebral Ischemia by Two-Photon Microscopy

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Free PMC article

In Vivo Imaging of Activated Microglia in a Mouse Model of Focal Cerebral Ischemia by Two-Photon Microscopy

Seoyeon Bok et al. Biomed Opt Express. .
Free PMC article

Abstract

Microglia are brain resident macrophages rapidly responding to various stimuli to exert appropriate inflammatory responses. Although they have recently been exploited as an attractive candidate for imaging neuroinflammation, it is still difficult to visualize them at the cellular and molecular levels. Here we imaged activated microglia by establishing intracranial window chamber (ICW) in a mouse model of focal cerebral ischemia by using two-photon microscopy (TPM), in vivo. Intravenous injection of fluorescent antibodies allowed us to detect significantly elevated levels of Iba-1 and CD68 positive activated microglia in the ipsilateral compared to the contralateral side of the infarct. We further observed that indomethacin, a non-steroidal anti-inflammatory drug significantly attenuated CD68-positive microglial activation in ICW, which was further confirmed by qRT-PCR biochemical analyses. In conclusion, we believe that in vivo TPM imaging of ICW would be a useful tool to screen for therapeutic interventions lowering microglial activation hence neuroinflammation.

Keywords: (000.1430) Biology and medicine; (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy.

Figures

Fig. 1
Fig. 1
ICW set up and microglia in a mouse model of focal cerebral ischemia. (a) Surgical procedures demonstrating ICW implantation. ICW was approximately 2 - 3 mm in diameter (black line). (b) Immunostaining of the brain at 24 hr post MCAO for Iba-1 (red) and CD68 (green). Nuclei are shown with DAPI (blue). IC and P represent ‘ischemic core’ and ‘penumbra’ regions, respectively. (c) FACS analysis of Iba-1 and CD68 expression levels in the ipsilateral side of the brain harvested from MCAO (pink line) or sham (black line) mice. Brain samples were pooled from a group of 4 mice. (d) In vivo TPM imaging of ICW for microglia using Iba-1 antibodies at 6 hr after sham (Visualization 1) or MCAO (Visualization 2) surgeries. Note that GFP-positive cells (green) in the brain are observed in both groups of mice, whereas Iba-1-positive microglia (red) were only detected in mice undergone MCAO. Scale bars in (b) and (d) indicate 100 μm.
Fig. 2
Fig. 2
In vivo imaging of activated microglia in ICW of MCAO mice by TPM. (a) A representative picture showing ICW implanted in the ipsilateral or contralateral side of mice subjected to the left MCAO. (b) TPM images for activated microglia in ICW implanted either in the ipsilateral (Visualization 3) or contralateral side (Visualization 4) of the ischemic infarct (n = 3 for ipsilateral; n = 2 for contralateral). Mice were administered with an antibody cocktail containing PerCP-Iba-1 (red) and AMCA-CD68 (blue) immediately prior to the TPM imaging. Scale bars indicate 100 μm. (c) Quantification of Iba-1 and CD68 expression levels in (b) at the ipsilateral (open bars) or contralateral side (closed bars). Data are the mean ± SEM (n = 3 for ipsilateral; n = 2 for contralateral) with * and ** indicate P < 0.05 and 0.01, respectively.
Fig. 3
Fig. 3
Increased cerebrovascular permeability in the ipsilateral side of focal cerebral ischemia. (a) Representative images of the brain perfused with FITC-lectin (n = 2). Note that a significant amount of FITC-lectin was extravasated in the ipsilateral side compared to the contralateral side of the infarct. (b) Evans blue extravasation in the ipsilateral (Ipsi) or contralateral (Contra) side at 24 hr post-MCAO. Data are the mean ± SEM for triplicate determinations with ** indicating P < 0.01 (n = 2 per group). (c) Immunostaining images of CD68-positive activated microglia in the ipsilateral or contralateral side of MCAO mice (n = 6 per group). Nuclei are shown with DAPI (blue). Scale bars in (a) and (c) denote 100 μm.
Fig. 4
Fig. 4
Indomethacin significantly attenuates microglial activation in ICW. (a) TPM images of activated microglia in ICW-bearing MCAO mice treated with either vehicle (Visualization 5) or indomethacin (Indo; Visualization 6) (n = 3 for vehicle; n = 4 for indomethacin). Imaging was performed at 24 hr post MCAO. Results are a representative image obtained from at least two independent areas per mouse, two animals per group. Scale bars denote 100 μm. (b) Quantification of Iba-1 and CD68 expression levels in MCAO mice treated with vehicle or indomethacin in (a). Data are the mean ± SEM with *** indicating P < 0.001 (n = 3 for vehicle; n = 4 for indomethacin). (c) mRNA expression levels in microglia isolated from MCAO or sham mice by FACS. (d) mRNA expression levels in microglia obtained from MCAO mice treated with vehicle or indomethacin (Indo) by FACS.

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