In vivo imaging of activated microglia in a mouse model of focal cerebral ischemia by two-photon microscopy

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. © 2015 Optical Society of America OCIS codes: (000.1430) Biology and medicine; (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy. References and links 1. A. Nimmerjahn, F. Kirchhoff, and F. Helmchen, “Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo,” Science 308(5726), 1314–1318 (2005). 2. H. Kettenmann, U. K. Hanisch, M. Noda, and A. Verkhratsky, “Physiology of microglia,” Physiol. Rev. 91(2), 461–553 (2011). 3. F. M. Lartey, G. O. Ahn, B. Shen, K.-T. Cord, T. Smith, J. Y. Chua, S. Rosenblum, H. Liu, M. L. James, S. Chernikova, S. W. Lee, L. J. Pisani, R. Tirouvanziam, J. W. Chen, T. D. Palmer, F. T. Chin, R. Guzman, E. E. Graves, and B. W. Loo, Jr., “PET imaging of stroke-induced neuroinflammation in mice using [18F]PBR06,” Mol. 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Introduction
Microglia are resident macrophages in the brain, and typically exist in the resting state characterized by ramified morphology [1].In response to brain injuries such as ischemic stroke, they are rapidly activated undergoing morphological changes to amoeboid morphology and displaying several activation markers including CD68 and major histocompatibility complex (MHC) class II [2,3].Activated microglia exert various immune functions including phagocytosis of dead cells and production of many cytokines, reactive oxygen, and nitrogen species [4,5].Although it is not yet clear whether these processes mediated by activated microglia are detrimental or beneficial in ischemic stroke, chronically activated microglia have been reported to exacerbate neurological injury [6] leading to the development of depressive-like behavior [7].Given their critical roles in contributing neuroinflammation thereby exacerbating the progression of various brain diseases including stroke [8], Alzheimer's disease, and Parkinson's disease [9], activated microglia have recently been exploited as an attractive candidate for non-invasive imaging to monitor neuroinflammation.Studies have reported temporal dynamics of neuroinflammation in mice and humans by PET imaging for 18 kDa translocator protein (TSPO), the outer mitochondrial membrane cholesterol transporter largely expressed in activated microglia [3,10].While these studies have demonstrated a direct clinical application for PET imaging of activated microglia, it is still difficult to visualize them at the cellular and the molecular levels.
Here we report in vivo imaging of activated microglia by establishing intracranial window chamber (ICW) in a mouse model of focal cerebral ischemia by using two-photon microscopy (TPM).Since its first demonstration in 1990, TPM has been applied to many in vivo studies of various organs including the brain with its advantageous features of higher imaging depths with minimal phototoxicity compared to other 3D microscopy techniques [11][12][13].In this report, we administered fluorescent labeled antibodies to wild-type mice subjected for focal cerebral ischemia and found that significant levels of Iba-1 and CD68 positive activated microglia were observed in the ipsilateral compared to the contralateral side of the infarct and that indomethacin significantly lowered CD68 signals in TPM imaging.We believe that in vivo imaging of ICW coupled with TPM would be a useful tool to better understand cellular and molecular processes involved in neuroinflammation.

Animals and intracranial window chamber (ICW)
8-week-old C57Bl/6 female mice were maintained in a germ-free environment and had access to food and water ad libitum.All animal procedures were approved by Institutional Animal Care & Use Committee at POSTECH.To establish ICW, 2 -3 mm diameter of left parietal skull of a female mouse was exposed and drilled using a high-speed dental drill with metal cutting burr tip (Diamond burr; Komet).The drilled skull was carefully removed and a sterile cover glass (3 mm diameter, Deckglaser) was placed on the dura and glued with mixture of acrylic powder (GC Fuji I).

Mouse model of middle cerebral artery occlusion (MCAO)
MCAO procedure was described as previously [3].Briefly a silicon suture (Doccol Corporation, MA) occluded the perfusion in the left MCA for 60 min, followed by removal of the suture and terminal ligation of the left carotid artery.Sham animals were similarly ligated for the left carotid artery.Blood perfusion before or during occlusion was measured by using Laser Doppler (PIM3, PERIMED) in the anesthetized mice that had been pre-warmed to the core body temperature of 38 °C on a heating plate measured by a rectal temperature probe (Physitemp).

Two-photon microscopy (TPM)
A commercial TPM system (TCS SP5 II, Leica) with a Ti-Sapphire laser ( respectively.Emission light was spectrally resolved into 4 channels by using a set of longpass dichroic mirrors at 495 nm, 560 nm, and 620 nm.3D images were acquired by taking multiple x-y plane images with a stepwise increment of 2 μm in the z direction from ICW surface down to 250 μm depth with 25X objective lenses (HCX IRAPO L25x, 0.95NA, water immersion, Leica).Excitation laser power was approximately 10 mW on the ICW surface, and increased linearly with depth in order to maintain the signal to noise ratio with which the excitation laser was manually calibrated before data acquisition by Z-compensation called 'Linear by AOTF (acousto-optical tunable filter)' mode equipped in LAS AF Lite software (Leica): The transmittance gain of excitation laser was then set to 30% at the beginning and was increased linearly as we go deeper in the in vivo brain tissues.Imaging speed was 0.78 frames per second and acquired images were processed by using LAS AF Lite software (Leica).The 3D volume images of 2 μm in depth at the stepwise increment were reprocessed to a projected image as a z-stack image.Each image of the z-stack (125 images in total (surface to 250 μm depth), 2 μm in thickness each) was then made to be composed of 512 by 512 pixels, in which each pixel had the intensity ranging from 0 to 4095 (12 bit).We then calculated the mean values for the pixel intensity in every pixel (512 x 512 pixels) of the every image of z-stack (approximately 100 images for rendering of microglia) for quantification by MATLAB.This quantification was performed in one region of interest (ROI), where we chose at least 3 different ROIs per mouse, totaling 2 ~4 mice per group.

Antibodies for TPM imaging
Iba-1 (Abcam) and CD68 (Abcam) antibodies were conjugated with PerCP and AMCA, respectively, by using EasyLink antibody conjugation kits (Abcam).Mice bearing ICW were intravenously injected with either PerCP Iba-1 alone or the mixture of PerCP Iba-1 and AMCA CD68 antibodies, immediately prior to in vivo TPM imaging.

Drug administration
Indomethacin (Sigma-Aldrich) was dissolved in 5% bicarbonate in water and administered intraperitoneally (10 mg/kg) at 0 and 6 hr post-MCAO to ICW-bearing mice.Control animals were similarly administered with vehicle (5% bicarbonate).

Fluorescent activated cell sorting (FACS)
Brain samples were pooled from a group of at least 4 mice.Left hemisphere of the brain was harvested from mice undergone sham or MCAO and digested in enzyme cocktail consisting pronase (Calbiochem), collagenase (Worthington), and DNase I (Sigma-Aldrich) for 30 min at 37 °C.Digested brain was filtered through 70 μm nylon mesh (BD Bioscience) and introduced to Percoll (GE Healthcare) gradient (30%, 37% and 70% Stock Isotonic Percoll balanced with Hanks balanced salt solution (Life technologies)).The microglia enriched at 70% -37% interphase were then collected and stained with Iba-1 or CD68 antibodies as described in immunostaining procedure above.Finally cells were resuspended in PBS + 3% fetal bovine serum (Life technologies) containing propidium iodide and analyzed by BD LSR II (BD Biosciences) or sorted by MoFlo XPD (Beckman Coulter).

Quantitative real time-polymerase chain reaction (qRT-PCR)
Total mRNA was isolated from FACS purified microglia using RNeasy mini kit (QIAGEN) following procedures according to the manufacturer's protocol.cDNA was synthesized using the following reagents: RNase-free DNase I (Promega), SUPERasein (Ambion), EDTA (Promega), dNTP (Invitrogen), random primers (Invitrogen), and Reverse Transcriptase (Promega).Synthesized cDNA was then subjected to PCR amplification using SYBR GREEN (Applied Biosystems).mRNA levels were calculated by relative quantification using comparative threshold cycle values based on those of β-actin according to the manufacturer's instructions (Applied Biosystems).

Immunostaining
Mice were cardiac perfused with 4% paraformaldehyde (PFA) (Dea Jung Chemicals) in PBS and the brain was harvested and made frozen sections, followed by fixation using 100% methanol for 30 min at the room temperature.The sections were incubated with 0.5% Triton X-100 in PBS for 5 min, followed by incubation with Iba-1 (goat anti-mouse Iba-1 polyclonal antibodies, Abcam) and CD68 primary antibodies (rat anti-mouse CD68 monoclonal antibodies, Abcam) for overnight at 4 °C.Secondary antibodies were anti-goat Alexa 546 (Life technologies) and anti-rat Alexa 488 (Life technologies), respectively, and incubated for 1 hr at room temperature.The sections were finally mounted with ProLong Gold antifade reagent with DAPI (Life technologies), and examined with a Zeiss Axio Scope with EC PLAN NEOFLUAR at 10 × , 20 × , and 40 × objective lenses.Digital images were taken using AxioCam HRM camera and processed with AxioVision 4.8 software.

Fluorescein isothiocyanate (FITC)-lectin infusion
Fluorescein labeled Lycopersicon esculentum (tomato) lectin (FITC-lectin) (Vector Lab) was dissolved in saline at 1 mg/ml concentrations and 0.1 ml was injected intravenously to mice.Mice were sacrificed within 5 min after injection by cervical dislocation and the brain sections were prepared as frozen sections, followed by post-fixation with ice-cold methanol for 10 min then finally mounted with ProLong Gold antifade reagent with DAPI.Images at 20 × objective were taken from the ipsilateral or contralateral side at two animals per group.

Evans blue extravasation
24 hr after MCAO, mice were intravenously injected with Evans blue (Sigma-Aldrich) at 30 mg/kg prepared in saline. 2 hr later, mice were perfused with acidified PFA (1% PFA (Dea Jung Chemicals) in 0.05 M citrate (Sigma-Aldrich) buffer at pH 3.5) for 4 min at 120 mmHg pressure to flush out all intravascular dye.Brains were then removed and ipsilateral and contralateral sides of the MCAO were placed into separate microcentrifuge tubes.Evans blue dye extraction from the tissues was then performed by using N,N-dimethylformamide (Sigma-Aldrich) and centrifugation at 15,000 g for 30 min.Supernatants were collected and optical density (OD) per g of tissue was measured by spectrophotometry.

Statistical analysis
Statistical comparisons of the data sets were performed by unpaired Student's t-test using Prism software (Version 4.00; GraphPad Inc.).The data were considered significantly different when P < 0.05.

Visualizing activated microglia in a mouse model of focal cerebral ischemia
With our previously reported MCAO techniques inducing focal cerebral ischemia in mice [3], we combined ICW techniques in these animals to perform in vivo TPM imaging [Fig.1(a)].We first examined ICW in mice ubiquitously expressing green fluorescent protein (GFP) and observed numerous GFP-positive cells in ICW at 6, 24, and 48 hr following either sham or MCAO procedures by using TPM.
We next sought for microglial markers that can be used in in vivo TPM imaging.To do this, we first performed immunostaining of the brain from mice undergone MCAO procedures to stain microglia and activated microglia by using Iba-1 and CD68 antibodies, respectively.Iba-1, ionized calcium-binding adapter molecule-1, is known to be specifically expressed in microglia but not in neurons, astrocytes or oligodendrocytes [14].CD68, also known as macrosialin is a lysosome-associated membrane glycoprotein whose expression is restricted to macrophage lineage, especially those activated microglia present in neurodegenerative diseases [15].We observed that there was a strong co-localization for Iba-1 and CD68 markers in the ischemic core region, while the adjacent penumbra demonstrated fewer areas of co-localization [Fig.1(b)].We further confirmed these findings by FACS and observed a significantly higher expression of both Iba-1 and CD68 in microglia isolated from mice challenged with MCAO compared to those with sham operation [Fig.1(c)].
Injection of PerCP-Iba-1 antibodies in GFP mice revealed a strong Iba-1 signal in ICW of mice undergone MCAO but not in sham animals by TPM [Fig.1(d)], likely to be due to the intact blood brain barrier in the latter.A number of those Iba-1-positive microglia observed in ICW of MCAO mice were in the ramified morphology, consistent with the immunostaining results [Fig.1(b)], indicating that they were microglia.These results therefore demonstrate that Iba-1-positive microglia can be detected in ICW of mice with the focal cerebral ischemia by TPM.We then injected CD68 antibodies together with Iba-1 antibodies in MCAO mice bearing ICW in order to detect activated microglia by TPM.In this experiment, we set up ICW either in the ipsilateral or the contralateral side of the infarct [Fig.2(a  Blood Brain Barrier is often being disrupted following cerebral ischemia resulting not only in the influx of leukocytes such as neutrophils, monocytes, and lymphocytes, but also in edema and cerebral hemorrhage, all of which may further contribute to the vicious cycle of ischemic brain injury [16,17].To test a possibility where the reduced CD68 signals in ICW at the contralateral side of the infarct in [Fig.2] were due to an intact blood brain barrier, we examined cerebrovascular permeability in these mice.We observed that there was a notable extravasation of intravenously administered FITC-lectin in the ipsilateral side of the infarct [Fig.3(a)], whereas the contralateral side demonstrated non-leaky blood vessels labeled by FITC-lectin [Fig.3(a)].Moreover, Evans blue dye extravasation assay revealed similar results where we observed significantly higher levels of Evans blue dye extracted from the ipsilateral compared to the contralateral side of the infarct [Fig.3(b)].By performing immunostaining, we confirmed that there were increased numbers of CD68-positive activated microglia in the ipsilateral side, while much fewer CD68-positive cells were observed in the contralateral side [Fig.3(c

ICW system by TPM can be utilized to monitor the efficacy of a therapeutic agent lowering microglial activation
To determine whether ICW imaging by TPM can be utilized to monitor the therapeutic efficacy of agent(s) lowering microglial activation, we tested indomethacin (Indo), a nonsteroidal anti-inflammatory drug, which has been previously reported to dampen microglial activation in a rat model of focal cerebral ischemia [15,18].By setting up ICW in the ipsilateral side of mice subjected to the left MCAO, we treated these mice with either indomethacin or vehicle at 0 (immediately after) and 6 hr post-MCAO and found that indomethacin significantly lowered Iba-1 and CD68 signals [Fig. 4  To investigate whether the above attenuated CD68 signal by indomethacin reflects decreased inflammation at the molecular level, we sorted microglia from these animals by FACS and performed qRT-PCR analyses against genes involved in macrophage inflammatory responses [19,20].We first observed that gene expressions including hypoxia-inducible factor-1α (Hif-1α), vascular endothelial growth factor (Vegf), interleukin-1β (Il-1β), and tolllike receptor-2 (Tlr-2) were significantly increased in microglia isolated from MCAO mice compared to those from sham animals [Fig.4(c)].Upon sorting activated microglia from MCAO mice treated with vehicle or indomethacin, we found that these increased in gene expressions were significantly reduced by indomethacin treatment [Fig.4(d)].Other genes examined such as S100 calcium binding protein A8 (S100A8), nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB), and chemokine (C-C motif) ligand 2 (Ccl2) were also decreased by indomethacin (data not shown).These results overall suggest that ICW imaging by TPM can be effectively utilized to monitor anti-inflammatory effects of a drug and that activated microglial imaging can reflect in situ inflammation at the molecular level.

Discussion
Here we report in vivo imaging of activated microglia in a mouse model of focal cerebral ischemia by using ICW combined with TPM.Because ischemic stroke triggers a complex cascade of cellular and molecular changes in many cell types, it is of great interest nowadays to understand cell-cell interactions [21], and to dissect and modulate immune dynamics (for example by inducing M2 polarized microglia) in order to better protect neurons from harmful immune responses associated with ischemia [22].TPM imaging has allowed to study various functional aspects of the brain at the cellular and molecular level including imaging analyses of 1) the structure and function of dendritic spines [23][24][25]; 2) networks of cortical vasculature [26,27]; 3) the structural dynamics of immune cells such as microglia [28].However, despite the powerful information that TPM can provide, there are only very few studies available on TPM imaging for activated microglia in rodent models of focal cerebral ischemia.The major limitation for the use of TPM to study such complex cell-cell interactions is perhaps to be due to its reliance on the availability of scarce and expensive transgenic animals.Previous studies examining microglia by TPM have been utilized CX3CR1 GFP/+ transgenic mice where CX3CR1 is expressed in microglia thereby labeled in green.By utilizing such system, Li and colleagues [29] have demonstrated in a parabiosis pair consisting wild-type and the transgenic mice undergoing photothrombosis stroke procedure that the expansion of activated microglia near the ischemic infarct was mainly due to proliferation of resident microglia rather than the infiltrating peripheral myeloid cells.Another study by TPM in combination with immunostaining has demonstrated that CX3CR1 can regulate morphological changes and surface expression of activated microglial markers including CD11b and CD68 [30].
Our study differs from others such that we have utilized wild-type animals and imaged activated microglia using TPM by intravenously injected fluorescent antibodies targeting Iba-1 and CD68 antigens.It has been previously reported that immunostaining results against CD11b, CD68 [31], and Iba-1 [32] are consistent with PET findings using TSPO radioligand in terms of the degree and location of activated microglia thus labeling for neuroinflammation.We believe that our ICW system labeling Iba-1 and CD68 positive activated microglia can also be utilized as a useful tool to monitor neuroinflammation.Furthermore, our results demonstrating that indomethacin can significant lower CD68 signal in ICW, accompanied by attenuation of proinflammatory gene expression at the molecular levels further validate our ICW system.In conclusion, we suggest that our ICW is easy to set up, there is no need to use scarce and expensive transgenic mice, ethically valuable as there is no need to sacrifice animals for time-consuming immunostaining, and there are diverse molecular targets to choose to do TPM imaging.This system may be potentially useful for developing and screening for therapeutic agents lowering microglial activation hence neuroinflammation.

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.
)].We observed that Iba-1 and CD68 signals were much stronger in the ipsilateral side than the contralateral side of the infarct [Fig.2(b) and 2(c)].

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.
)], the results in good agreement with ICW by TPM above [Fig.2(b) and 2(c)].These results overall suggest that the strong microglial activation by Iba-1 and CD68 signals in the ipsilateral side of ICW by TPM is resulted from the disrupted blood brain barrier caused by focal cerebral ischemia.

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.