As brain-resident immune cells, microglia (MG) survey the brain parenchyma to maintain homeostasis during development and following injury. Research in perinatal stroke, a leading cause of lifelong disability, has implicated MG as targets for therapeutic intervention during stroke. Although MG responses are complex, work in developing rodents suggests that MG limit brain damage after stroke. However, little is known about how energy-limiting conditions affect MG survival and mobility (motility and migration) in developing brain tissues. Here, we used confocal time-lapse imaging to monitor MG viability and mobility during hypoxia or oxygen-glucose deprivation (OGD) in hippocampal tissue slices derived from neonatal GFP-reporter mice (CX3CR1^GFP/+). We found that MG remain viable for at least 6 h of hypoxia but begin to die after 2 h of OGD, while both hypoxia and OGD reduce MG motility. Unexpectedly, some MG retain or recover motility during OGD and can engulf dead cells. Additionally, MG from younger neonates (P2–P3) are more resistant to OGD than those from older ones (P6–P7), indicating increasing vulnerability with developmental age. Finally, transient (2 h) OGD also increases MG death, and although motility is rapidly restored after transient OGD, it remains below control levels for many hours. Together, these results show that MG in neonatal mouse brain tissues are vulnerable to both transient and sustained OGD, and many MG die within hours after onset of OGD. Preventing MG death may, therefore, provide a strategy for promoting tissue restoration after stroke. © 2012 Wiley Periodicals, Inc.

INTRODUCTION

Stroke is a leading cause of disability and death in the aged population but is also common in newborns. For example, cerebrovascular accidents occur in 20–30% of infants born at less than 35 gestational weeks (Derugin et al., 2000; Yager and Thornhill, 1997). These ischemic events often cause long-term, debilitating conditions including cerebral palsy, seizures, and cognitive disorders (Nelson, 2007). A better understanding of the cellular basis of brain responses to stroke could lead to more effective treatments in preterm and term infants.

Classic stroke research focusing on the effects of energy depletion on neurons is being complemented by an emerging glio-consciousness that recognizes roles for glial cells in the progression and potential recovery from stroke. As mediators of inflammation, brain-resident microglia (MG) have been thought to exacerbate ischemic injury (Deng et al., 2011; Lai and Todd, 2006; Wu etal., 2012; Yenari et al., 2010). However, several lines of evidence from experimental ischemic models suggest that MG can improve stroke outcome by supporting tissue homeostasis, rapidly clearing dead cells, and secreting trophic factors that promote cell survival and regeneration (Hanisch and Kettenmann, 2007; Weinstein et al., 2010). For example, depleting endogenous MG in adult (Lalancette-Hebert et al., 2007) or neonatal (Faustino et al., 2011) brain in vivo, or in ex vivo slices (Montero et al., 2009), worsens stroke outcome. Conversely, introduction of exogenous MG improves stroke outcome in vivo (Hayashi et al., 2006; Imai et al., 2007; Kitamura et al., 2004, 2005) and in ex vivo preparations (Neumann et al., 2006). Moreover, MG can reduce neurotoxic effects in ex vivo slices by engulfing neutrophils (Neumann et al., 2008) and releasing growth factors and cytokines, including TNF-α, which protect neurons (Lambertsen et al., 2009). These studies indicate that MG may play important roles in tissue repair after stroke.

Although it is clear that ischemia induces rapid MG activation in the neonatal brain (Ivacko et al., 1996), there is little information on its consequent effects on MG survival. Studies using cultured rodent MG report different levels of cell death (15–88%) following simulated ischemia (Lyons and Kettenmann, 1998; Yenari and Giffard, 2001). However, such in vitro studies are complicated by the possibility that cultured cells may develop resistance to hypoxia (Chock and Giffard, 2005). To date, MG viability and mobility during ischemic conditions in neonatal brain tissues have remained largely uninvestigated.

To address these questions, we used oxygen-glucose deprivation (OGD) as a model of stroke in hippocampal slices from neonatal GFP-reporter mice. Confocal time-lapse imaging enabled us to monitor MG mobility and survival during and following energy-limiting conditions. We show that MG in these tissues respond to stroke-like conditions in diverse ways: some MG are resistant and seem unaffected, others show a transient or sustained reduction in cell mobility, and many MG die.

MATERIALS AND METHODS

Animals and Preparation of Tissue Slices

For all experiments, we used heterozygous mice expressing GFP under control of the fractalkine receptor promoter (CX3CR1^+/GFP; Jung et al., 2000). GFP is expressed in parenchymal MG as well as in perivascular and meningeal cells, which are easily distinguishable from MG. We cannot rule out possible subtle effects on MG mobility in CX3CR1^+/GFP mice. However, wild-type and CX3CR1^+/− mice show similar infarct volumes following ischemia (Denes et al., 2008). Acutely isolated hippocampal slices were prepared from neonatal (P5–P7 unless otherwise stated) mice as detailed previously (Kurpius et al., 2007). Briefly, mice were swiftly decapitated, and brains were removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 124; KCl 3; NaH₂PO₄ 1.3; MgCl₂ 3; HEPES 10; CaCl₂ 3; glucose 10. Excised hippocampi were cut transversely (400 μm thick) using a manual tissue chopper (Stoelting) and maintained in aCSF. Animals were used in accordance with institutional guidelines, as approved by the animal care and use committee.

Hypoxia and Oxygen-Glucose Deprivation

Oxygen deprivation (hypoxia) was achieved by bubbling N₂ into aCSF for 15 min. Oxygen and glucose deprivation (OGD) was achieved by replacing 10 mM glucose with 10 mM sucrose and bubbling N₂ into aCSF for 15 min. Sometimes, Sytox Orange (Molecular Probes, #S11368) or Topro-3 (Molecular Probes, #T3605) were added to aCSF at 1:10,000 before N₂ bubbling. Only cells whose membranes are compromised (leaky) are labeled with Sytox or Topro-3. For each experiment, 4–10 mL of media was bubbled, warmed briefly in a 37°C water bath, and then immediately applied to previously mounted slices in a chamber that was then sealed for imaging. In transient OGD experiments, sealed chambers were maintained for 2 h at 37°C before subsequent mounting and imaging.

Drug Treatments

Apyrase (Sigma Aldrich, no. A6132), an enzyme with both adenosine 5′-diphosphatase and -triphosphatase activity, and ATP (Sigma, no. FLAAS) were used at 200 U/mL and 0.5 mM in aCSF, respectively.

Time-Lapse Confocal Imaging

Four to twelve acutely excised tissue slices were mounted in a custom-built chamber containing ∼3 mL aCSF. The sealed chamber was placed on the microscope stage and warmed to ∼35°C by continuous, gentle warm air (Kurpius et al., 2007). Fluorescence images were captured using a Leica SP5 MP confocal/multiphoton imaging system with a xyz motorized stage on an upright platform. The following probes were imaged with the indicated laser lines: GFP (Argon 488 nm), Sytox Orange (HeNe 543 nm), and Topro-3 (HeNe 633 nm). The pinhole typically was opened to two Airy disc units to improve light collection and signal-to-noise. The chamber media was not changed during the course of imaging, except when performing transient OGD experiments or upon addition of drug. Pilot experiments showed that, under control conditions with 12 slices in a chamber, neonatal MG remain actively motile for at least 8 h of imaging without any media change. To capture a large (775 μm × 775 μm) field of view containing 40–60 MG, images were collected using a 20×/0.7 Plan Apo objective lens at a resolution of 1.4 pixels/μm. A typical time-lapse imaging experiment captured 15 optical planes at 3-μm z-step intervals spanning 45–60 μm in the axial dimension from the slice surface. Stacks of images were captured at 3–10-min intervals. Multisite imaging allowed us to image multiple tissue slices from separate littermate animals simultaneously under identical conditions. Imaging sessions typically commenced about 30 min after tissue slicing and lasted several hours.

Image Processing

Images were collected and collated using Leica LAS AF software. Image stacks were assembled using Leica LAS AF software or ImageJ (Wayne Rasband, NIH). All images were processed using the “Smooth” filter in ImageJ to reduce noise. Comparisons were made on images processed identically. All movies generated represent the same xyz tissue volume, although they may differ in lengths of time.

Analysis of Microglial Cell Death

MG cell death events were identified by concomitant loss of GFP and gain of Sytox. Manual counts of only GFP-expressing parenchymal MG were made for the duration of a time-lapse sequence for each field of view analyzed. Because a few cells moved in or out of focus, the total number of MG was calculated as the sum of the number of GFP-positive MG at the end of the experiment (which represents the number of living MG) plus the number of dying MG counted during the sequence. Percent cell death was calculated as follows: (no. of dead MG cells/total no. of MG cells) × 100.

Analysis of Microglial Motility

We developed a simple measure of MG cell motility using ImageJ. First, 3D image stacks were combined to make 2D projection images for each time point. Next, to account for any x–y tissue drift, 2D projection images were registered using the StackReg plugin (Thevenaz et al., 1998). Registered images were smoothened to reduce background noise. To define the cell boundary, an arbitrary threshold was applied uniformly to all images in a given time sequence. To generate difference images, the absolute difference between two sequential thresholded images in a time series was calculated using the “Difference” tool of the “Image Calculator” feature of ImageJ (see Supp. Info. Fig. 1). Sequential difference images in a time sequence (i.e., images 1 and 2) were used to generate a motility index (MI), which is a percent change in area calculated as follows:

Details are in the caption following the image — **Figure 1**
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OGD induces microglial cell death in acutely isolated neonatal hippocampal slices. A: Time-lapse imaging shows that many nonmicroglial cells die under control conditions, as shown by Sytox labeling of nuclei of non-GFP expressing cells. Several dying cells are evident within the first 2 h after tissue excision. More nonmicroglial cells die during the next several hours (arrows and arrowhead), but no GFP-expressing MG die (assessed by loss of GFP and gain of Sytox) during this period of time. B: Images from a time-lapse movie of a MG cell that dies during OGD. The MG cell loses GFP and within minutes becomes labeled with Sytox. Note that the cell loses GFP before showing obvious signs of blebbing. See Supp. Info. Movie 1. C: Quantification shows that, for the dying MG in (B), the GFP signal rapidly decreases in the soma as Sytox signal subsequently increases in the nucleus. **D–D′:** Some MG become unhealthy looking after prolonged OGD (D′), showing rounded soma (arrow) and beaded branches (arrowheads), but retain GFP and fail to take up Sytox (red). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Statistical Analysis

Data from several slices and different animals were pooled and analyzed. Each animal represents a separate experiment. All results are reported as mean ± standard deviation of the population. Statistical significance was assessed using Student's t test at the significance level P < 0.05.

RESULTS

Prolonged OGD But Not Hypoxia Induces MG Death in Developing Brain Tissue Slices

Microglia (MG) are viable and sustain high levels of mobility (motility and migration) in acutely isolated neonatal hippocampal slices (Kurpius et al., 2006, 2007; Stence et al., 2001). Therefore, we used this preparation to study effects of stroke-like conditions on MG mobility and viability in neonatal tissues. To assess the vulnerability of MG to hypoxic-ischemic injury, we used two-channel time-lapse confocal imaging of slices from GFP-reporter mice bathed in Sytox-containing aCSF. Sytox is a membrane impermeable DNA-binding dye that labels only dead and dying cells with compromised membranes. MG in these mice constitutively express GFP and can be easily identified. A progressive loss of GFP can be used to detect MG death as it occurs in real time. We focused our analyses in hippocampal area CA1, because this area is especially vulnerable to ischemia (Lehotsky et al., 2009).

Under control conditions, many non-MG cells (lacking GFP) in acutely isolated neonatal (P5–P7) slices take up Sytox during the first several hours of imaging (Dailey and Waite, 1999), presumably as a result of traumatic injury induced by tissue slicing (Fig. 1A arrows and arrowhead). However, under these control conditions, we never observed MG cell death as determined by loss of GFP and subsequent Sytox uptake (n = 740 MG cells, 14 slices, three animals).

When slices were imaged continuously for 6 h under OGD, we found that many MG rapidly lost GFP signal (within 5–10 min) and subsequently gained Sytox (Fig. 1B,C and Supp. Info. Movie 1). However, some unhealthy looking MG with beaded branches and rounded soma retained GFP and failed to take up Sytox throughout the 6 h of OGD (Fig. 1D,D′). Thus, we construed loss of GFP signal as an indicator of loss of membrane integrity coinciding with MG cell death.

MG death events were frequently observed during severe energy depleting conditions (Fig. 2A). Under hypoxia, MG death was infrequent and not significantly higher than control conditions. We observed only 1.1 ± 1.8 MG cell death events per field of view (range, 0–7) during 6 h of hypoxia, amounting to 1.9% of MG (n = 24 slices from three animals; Fig. 2A,B). A little over half (14 of 24) of the slices had no MG death events. In contrast, during 6 h of OGD, the number of dying MG cells increased dramatically to 22.1 ± 3.8 per field of view (range, 14–27), corresponding to 44.0% of MG (n = 16 slices from three animals; Fig. 2A,B). This increased MG death was highly significant when compared with both control and hypoxic conditions. MG death rarely occurred before 2 h of OGD and increased over subsequent hours (Fig. 2C), peaking at about 5 h (Fig. 2D). Return to normal oxygen-glucose conditions after 6 h of OGD did not restore viability to unhealthy-looking MG (as in Fig. 1D′; data not shown). Taken together, our results show that neonatal MG survive during 6 h of hypoxia but begin to die within 2 h of OGD.

Both Hypoxia and OGD Inhibit MG Motility

MG motility is important for MG functions including regulation of synaptic remodeling, migration to sites of injury, and phagocytosis of dead cell debris (for review, see Parkhurst and Gan, 2010). These MG functions may be critical for tissue recovery after stroke. To investigate the effect of energy-limiting conditions on MG motility in neonatal tissues, motility was quantified by calculating changes in cell morphology in sequential time-lapse images to obtain a MI (see Methods section and Supp. Info. Fig. 1). Under control conditions, MG were continually motile, repeatedly extending and retracting processes for the duration of the imaging period (Fig. 3A, top row). In contrast, both hypoxia and OGD caused a persistent reduction in MG dynamism, especially in the stratumradiatum (SR) region (Fig. 3A, middle and bottom rows, respectively).

Quantitative analyses of MG motility over large fields of view containing tens of MG cells in the SR of area CA1 confirmed these qualitative impressions (Fig. 3B). Under control conditions, MG motility increased over the first couple of hours. Thereafter, MG motility was maintained at a high level for up to 6 h (n = 6 slices from three animals). Conversely, hypoxia (n = 6 slices from three animals) and OGD (n = 5 slices from two animals) caused a significant reduction in MG motility over the first 2 h of imaging, and this low level was maintained for the rest of the imaging period (Fig. 3B). When averaged over the duration of the 6 h imaging period, the mean MG MI was significantly lower in hypoxia- and OGD-treated slices relative to control (Fig. 3C). There was no significant difference between hypoxia- and OGD-treated slices (Fig. 3C and Supp. Info. Movie 2), indicating that MG viability and motility are differentially affected by hypoxia and OGD.

Some MG Are Resistant to OGD

Although motility of most MG in the SR was inhibited by OGD, we noticed that some MG in the SR and in other layers—stratum pyramidale (SP) and stratum oriens (SO)—were resistant to OGD. Individual MG cells fell into one of three general categories of motility behavior, which we termed Types 1, 2, and 3 (Fig. 4A). Type 1 MG cells were persistently motile throughout the imaging period. These cells showed a high level of process dynamism that was evident in difference images as large white patches around the cell margin (Fig. 4A, top row). Type 2 MG cells were initially quiescent but became motile sometime during imaging, either after initial branch retraction (e.g., during control conditions) or after a transient stall in process motility (e.g., during the early stages of hypoxic or OGD conditions; Fig. 4A, middle row). Finally, Type 3 cells showed little process dynamism throughout the imaging period (Fig. 4A, bottom row).

The percent of Type 1 MG dropped significantly from 91.1% during control conditions to 37.9% during hypoxia and 30.3% during OGD (Fig. 4B). The percent of Type 2 (transiently immotile) cells remained largely unchanged during hypoxia (8.9%) relative to control (9.9%), whereas it was halved during OGD conditions (4.7%). Conversely, the percent of Type 3 (immotile) MG increased from 0% under control conditions to 52.2% during hypoxia and 65.0% during OGD. Although the majority of MG were immotile during hypoxia and OGD, to our surprise, some MG remained active under these conditions, showing little or no inhibition of motility (47.8% during hypoxia and 35.0% during OGD). These OGD-resistant MG were usually located in the SO or SP (Fig. 4C). Time-lapse sequences showed that some MG that recovered motility or remained active during OGD could engulf nearby dead cells (Fig. 5 and Supp. Info. Movie 3). Thus, while energy-depleting conditions largely inhibit MG motility, a level of motility is retained or restored in some MG that enable them to engage and clear dead and dying cells.

Extracellular Purines Control OGD-Resistant MG Motility

During normoxic/normoglycemic conditions, MG motility and chemotaxis are strongly regulated by extracellular purines (Farber et al., 2008; Haynes et al., 2006; Honda et al., 2001). Therefore, we tested whether extracellular purines are necessary and sufficient to support residual MG motility observed during OGD. Application of apyrase (200 U/mL), an ATP/ADP-degrading enzyme, rapidly abolished any residual MG motility during OGD, confirming its dependence on endogenous extracellular purines (Fig. 6A,B and Supp. Info. Movie 4). Additionally, when ATP (0.5 mM) was applied to tissue slices during OGD, MG were able to extend branches and migrate in a directed manner toward the ATP source (Fig. 6C,D and Supp. Info. Movie 5). Taken together, these results indicate that OGD compromises mobility and viability for most MG in the neonatal hippocampus. However, a subset of OGD-resistant MG persists whose extracellular purine-dependent motility could facilitate their engagement with dead cells.

MG Viability and Mobility During OGD Differ Developmentally

Although earlier studies suggested that the immature brain is more resistant to ischemia than the mature brain, more recent studies have challenged this concept (Schaller, 2007). To determine whether MG viability and mobility are developmentally sensitive, we compared MG in slices from younger (P2–P3) and older (P6–P7) neonates. We found that MG in younger neonates are more resistant to OGD, based on several parameters. First, the number of dying MG was significantly lower in tissues from younger neonates (2.2 ± 1.5 events per field of view in 20 slices from three animals) in comparison with tissues from older neonates (20.9 ± 4.1 events in 11 slices from two animals). Because slices from younger mice had fewer MG, we determined the percent MG death and confirmed that this was also significantly lower in tissues from younger neonates (6.1% ± 4.2%) than older ones (42.7% ± 10.9%; Fig. 7A; P < 0.0005). Furthermore, the timing of earliest MG death was significantly later in tissues from younger (199 ± 26 min per slice) versus older (123 ± 40 min per slice) neonates (Fig. 7B; P < 0.0005).

The effects of OGD on MG mobility also differed with developmental age. The fraction of persistently motile (Type 1) MG decreased significantly from 82.2% ± 12.8% at P2–P3 to 19.4% ± 13.9% at P6–P7 (Fig. 7C; P < 0.0005). Also, we tracked the migration of surviving MG during OGD and found that the fastest MG in P2–P3 neonatal slices migrated at a significantly higher peak velocity (1.8 ± 0.4 μm/min per slice; n = 15 cells from five slices) than surviving MG in P6–P7 slices (0.34 ± 0.1 μm/min; n = 15 cells from five slices, Fig. 7D; P < 0.0005). Consequently, the total distance traveled by MG during 6 h of OGD dropped significantly from 857 ± 239 μm to 298 ± 74 μm as development progressed (Fig. 7E; P < 0.0005). The migration tracks of two representative cells in younger (P2) and older (P6) slices are shown (Fig. 7F,G and Supp. Info. Movie 6). These results indicate that MG are functionally more resilient to OGD in younger neonatal tissues than older ones.

Transient (2 h) OGD Has Lasting Effects on MG Mobility and Survival

Thus far, our energy-limiting conditions were applied continuously for several hours. However, hypoxic-ischemic episodes can result from transient blood vessel occlusion followed by reperfusion. Therefore, we investigated the effect of transient OGD (tOGD) on MG mobility and survival. We used a 2-h tOGD period, because we rarely observed MG death in neonatal (P5–P7) slices during this time period (Fig. 2C,D). The tOGD imaging protocol consisted of baseline recordings for 1 h, followed by recordings for 2 h during OGD, and concluding with 1 h of imaging during washout to mimic reperfusion.

As observed during sustained (6 h) OGD, we found that most MG became immotile during tOGD, but some maintained their motility throughout tOGD (Fig. 8). As noted earlier, cells in the SP usually remained motile during OGD (Fig. 8B, top row) while those in the SR became immotile (Fig. 8B, bottom row). For those MG that were inhibited by tOGD, motility was reduced within 15 min of OGD exposure and was subsequently maintained at low levels throughout the tOGD period. However, MG rapidly recovered upon washout. By 3 min of washout, motility of extant processes was restored, and within 15 min of washout, new MG processes were generated (Fig. 8B; see also Supp. Info. Movie 7). Quantitative analyses showed that the motility of persistently motile Type 1 MG (n = 12 cells from three slices) continued largely unchanged during the 4-h imaging period (Fig. 8C,D). However, MG that became immotile during tOGD had a lower MI to start with but recovered even beyond baseline levels after OGD (n = 12 cells from four slices; Fig. 8C,D). These observations indicate that MG in developing brain tissues can resist or rapidly recover from short-term (≤2 h) energy-limiting conditions.

Because our tOGD studies focused solely on the motile responses of MG during and immediately following the insult, we extended these studies to investigate possible longer-term effects of tOGD on MG mobility and viability. For these studies, hippocampal slices were isolated from neonates, and half were carried under normal oxygen and glucose levels (control) while the other half were subjected to tOGD. Both control and tOGD slices were then placed together in imaging chambers containing normal aCSF, and slices were imaged simultaneously by multisite time-lapse imaging for several hours starting at 4 h after the treatment period (Fig. 9A). Over this 6-h posttreatment period, motility indices for OGD-treated slices (n = 7 slices, two animals) were persistently lower than those for their control counterparts (n = 11 slices, two animals; Fig. 9B). tOGD reduced average motility levels to about two-thirds of normal for at least 6 h (Fig. 9B,C). Moreover, analysis of MG migration showed that the most motile MG cells traveled much shorter distances in slices previously subjected to tOGD (246 ± 56 μm) relative to MG in control slices (490 ± 234 μm; Fig. 9D).

Finally, we assessed MG viability and found a significant increase in the number of MG death events following tOGD (mean = 3.2 ± 1.9 events per field of view; 35 total events; range, 0–6; n = 11 slices, two animals) compared with control (mean = 0.17 ± 0.37; two total events in 12 slices from two animals) when assessed at 6 h after the end of tOGD (Fig. 9E; P < 0.0005). Taken together, these results suggest that even tOGD has persisting effects on MG mobility and viability in neonatal brain tissues.

DISCUSSION

We investigated MG viability and mobility (motility and migration) in developing mouse brain tissue slices during energy-limiting conditions in real time. We show that virtually all MG survive during 6 h of hypoxia, whereas nearly half of the MG die during 6 h of oxygen and glucose deprivation (OGD). Additionally, both hypoxia and OGD reduced the number of motile MG as well as the level of motile activity. However, some OGD-resistant MG persist and can phagocytize nearby dead cells. MG viability and mobility during OGD are higher in tissues from younger neonates relative to older neonates. Finally, we show that transient (2 h) OGD has long-lasting effects on MG viability and mobility.

Microglial Viability During OGD

MG cell death studies have been performed in cultured MG where varying degrees of susceptibility to 6 h of OGD were reported (Lyons and Kettenmann, 1998; Yenari and Giffard, 2001). However, cell metabolism may change during long-term cell culture, and the extent to which these results reflect cell behaviors in living tissues and in vivo has been unclear. MG have been found in the ischemic core 24 h after permanent middle cerebral artery occlusion (MCAO) in both the adult (Mabuchi et al., 2000) and neonatal (Wen et al., 2004) brain, suggesting that some MG may survive for some time under permanent ischemia. By 24 h after neonatal transient MCAO, MG density decreases by about 40% in the ischemic core relative to the penumbra (Faustino et al., 2011). However, clear indication of the degree of MG death in living brain tissues during ischemia is lacking. Indeed, it is difficult to estimate the fraction of surviving MG from such in vivo studies due to possible proliferation of surviving MG, migration from surrounding tissues, or infiltration of hematogenous cells of monocyte lineage.

We used an ex vivo neonatal acute brain slice system, because it retains the native cell and tissue organization, is easily accessible for imaging, and avoids potential problems associated with changes in cell metabolism in long-term cell and tissue-culture preparations, or infiltration and redistribution of cells in vivo. Using two-channel time-lapse imaging, we monitored MG viability in developing brain tissues and, in line with previous in vitro studies (Lyons and Kettenmann, 1998; Yenari and Giffard, 2001), showed that OGD but not hypoxia induces MG death. Furthermore, we show that MG begin to die after 2 h of OGD. MG in neonates were recently shown to help protect the developing brain during stroke (Faustino et al., 2011). In light of this, our observation of MG death during OGD and following transient OGD suggests that enhancing MG survival during ischemic injury could serve as a neuroprotective strategy for neonatal stroke.

To enhance MG survival, an understanding of the modes and mechanisms of MG death during OGD is critical. During ischemia, both necrotic and apoptotic modes of cell death have been reported (Mehta et al., 2007), and antiapoptotic treatment is protective in both adults (Fong et al., 2010; Lee et al., 2004; Li et al., 2012) and neonates (Carlsson et al., 2011; Nijboer et al., 2011). Whether MG death during OGD is necrotic, apoptotic, or necroptotic (Vandenabeele et al., 2010) has not been determined. However, during in vivo neonatal ischemia, MG reportedly activate an apoptosis executioner caspase, caspase-3 (Manabat et al., 2003; Ness et al., 2008; Peng et al., 2010). It is presently unclear whether this caspase-3 activation indicates commitment to apoptotic cell death, or simply MG activation that does not lead to death (Burguillos et al., 2011).

With regard to cell death mechanisms, other cell types are vulnerable to ischemic insult via calcium dysregulation, resulting in calcium overload (Szydlowska and Tymianski, 2010). Indeed, calcium overload induces MG death (Nagano et al., 2006), and this may contribute to MG death during OGD. Both glutamatergic and purinergic signaling have been shown to mediate excitotoxic death in neurons, astrocytes, and oligodendrocytes (Matute, 2011). Although, to the best of our knowledge, glutamate receptors are not expressed in neonatal MG, it has been reported that MG in the adult hippocampus express glutamate receptors a few days after transient ischemia (Gottlieb and Matute, 1997). However, glutamate failed to elicit electrophysiological or morphological responses in hippocampal (Wu and Zhuo, 2008) and retinal (Fontainhas et al., 2011) MG, suggesting that, in the resting/surveillant state, MG lack functional glutamate receptors. Thus, MG may only express glutamate receptors after some delay following activation and therefore may be less vulnerable to glutamate excitotoxicity during early stages of ischemia (Matute, 2011; Matute et al., 2006).

In contrast, it is well established that MG express several P2X- and P2Y-type purinergic receptors in the resting state. Recently, both the P2X4 and P2X7 receptors have been implicated in ATP-induced cell death in macrophages (Hanley et al., 2012; Kawano et al., 2012). Because these receptors are detected in MG as early as E16 (Xiang and Burnstock, 2005) and are upregulated in hippocampal MG in response to ischemia (Cavaliere et al., 2003, 2005), it is tempting to speculate that, with high levels of extracellular ATP during OGD (Dale and Frenguelli, 2009), purinergic receptor activation could mediate excitotoxic MG cell death. However, whether calcium influx induced by glutamate- or ATP-dependent signaling mediates OGD-induced MG death remains to be determined.

Microglial Motility During OGD

MG motility has been extensively studied previously (Ohsawa and Kohsaka, 2011; Parkhurst and Gan, 2010) and may monitor and remove synapses with reduced synaptic activity after ischemia (Wake et al., 2009). However, while several studies of MG motility have been performed in adult brain, motility in neonatal brain in vivo remains little studied under normal or pathological conditions. The present work is the first report on the effects of OGD on MG motility in neonatal brain tissues and shows that hypoxia and OGD inhibit MG motility.

Studies in adult CNS have differed with respect to effects of blood flow loss on MG motility. In the postmortem spinal cord of adult mice, MG motility is retained for a period of time independent of blood flow (Dibaj et al., 2010). However, in vivo imaging in the adult cortex showed that disruption of blood flow by photothrombosis, transient artery occlusion, or global ischemia abolished MG motility (Masuda et al., 2011). This latter study reported that restoration of blood flow after a 30-min occlusion restored motility. Although our results are consistent with Masuda et al. ( 2011) in that they show a reversible loss of MG motility, we extended these findings by showing that some neonatal MG regain mobility during OGD, albeit at reduced levels. The ability of MG to regain mobility after 2 h of OGD suggests that it may be possible to rescue MG function even after several hours of ischemia in vivo.

Paradoxically, in P11–P27 rat hippocampal slices, there is an increase in extracellular ATP during OGD and a burst of ATP release following OGD (Dale and Frenguelli, 2009). Because ATP acts as a MG chemotactic and chemokinetic signal (Davalos et al., 2005; Haynes et al., 2006; Honda et al., 2001), it is possible that increased extracellular ATP promotes MG motility after OGD. We showed that, despite an initial restoration of MG motility (Fig. 8C), it is significantly reduced several hours later (Fig. 9). This persistent reduction of motility may result from lower extracellular ATP levels after the initial ATP burst following transient OGD. Indeed, cultured astrocytes subjected to 2 h of transient OGD show a dramatic increase in ATP release during the first hour of washout, but ATP release drops significantly below control levels 5 h later (Iwabuchi and Kawahara, 2009). Similarly, “secondary energy failure” occurs in the neonatal brain 4–6 h into reperfusion after transient hypoxic-ischemic injury (Ten et al., 2010), which may also contribute to the reduced MG mobility. Together, these studies suggest that changes in both extracellular and intracellular ATP levels following stroke could regulate MG mobility.

OGD-Resistant MG

Although most MG were inhibited by OGD, we found that about 30% of MG in acute P5–P7 hippocampal slices resisted the mobility-inhibiting effects of OGD and either retained or regained motile activity during energy limiting conditions. The basis for this resistance is presently unclear, but “resistant” MG were frequently located near the pyramidal cell body layer (SP). It is possible that MG in close proximity to the neuronal cell body layer experience higher levels of metabolites that confer resistance to OGD. Both neurons and astrocytes release ATP during OGD (Dale and Frenguelli, 2009; Iwabuchi and Kawahara, 2009; Liu et al., 2008), and both astrocytes and MG participate in regenerative ATP-induced ATP release (Davalos et al., 2005; Dou et al., 2012). Therefore, higher extracellular purine levels near the SP may facilitate resistance of MG in this region during OGD. Indeed, we showed using apyrase that the persistence of motility in these MG during OGD is dependent on extracellular purines, further supporting the idea that residual MG motility is driven by extracellular purines.

Using time-lapse imaging, we showed that these OGD-resistant MG can engulf dead cells, thereby clearing the brain of potentially toxic cell debris (Neumann et al., 2009). This indicates that the phagocytic signaling machinery is intact in MG and can be harnessed. Because even limited phagocytosis by MG may be important in protecting the neonatal brain during stroke (Faustino et al., 2011), enhancing MG resistance to ischemic injury may be beneficial in stroke therapy.

Developmental Sensitivity of MG to OGD During the Neonatal Period

There is conflicting data as to whether the developing brain is more susceptible to ischemic injury than the adult brain (Schaller, 2007; Yager and Thornhill, 1997). We attempted to inform this debate on a cellular level by investigating MG susceptibility during the early neonatal period. Using dissociated neonatal cultures, Chock and Giffard ( 2005) showed that MG become less vulnerable to OGD with increasing time in culture. However, in acutely isolated slices, we found that MG viability and motility during OGD are better preserved in younger neonatal tissues. These differences may be due to differences in the preparations. Interestingly, previous work in rats reported similar resistance of neurons to hypoxia-ischemia in younger versus older neonates (Towfighi et al., 1997). Therefore, during the early postnatal period, MG, like neurons, become more vulnerable to ischemic injury as development progresses. It is possible that with age, MG become intrinsically more vulnerable to ischemic insult. However, cell extrinsic factors released in tissues by other cells, including neurons, could promote MG resistance to OGD. It will be important to determine what feature(s)—intrinsic and/or extrinsic—of the early postnatal period promotes MG resistance to OGD and if such resistance could enhance neuronal survival.

This is the first report on MG viability and mobility during stroke-like conditions in developing brain tissues. Both hypoxia and OGD inhibit MG motility, whereas OGD but not hypoxia induces MG death within the first few hours. The limiting effects of OGD on MG survival and mobility increase with age and some surviving MG can retain or recover motility and phagocytize debris, thereby contributing to tissue restoration. It remains unclear what specific factors regulate MG death during OGD, and future studies will need to address these questions. Nevertheless, these results extend our understanding of MG survival and behavior in developing brain tissues and suggest that survival of MG is an important consideration when developing therapeutic strategies for managing stroke in the perinatal brain.

Acknowledgements

The authors thank Ms. Leah Fuller for assistance with mouse colony management and genotyping. The authors declare that they have no conflict of interest.

Supporting Information

Additional Supporting Information may be found in the online version of this article.

Filename	Description
GLIA_22394_sm_SuppFig1.pdf152.3 KB	Supporting Information Figure 1. Method of generating ‘difference images’ for motility index analysis. First, confocal z-stacks for each time-point in a series were combined (maximum brightness) to obtain ‘projection images.’ The series of projection images was then ‘registered’ (using the StackReg plugin in ImageJ) to adjust for any lateral drift. For illustration purposes, images A and A′ represent two adjacent registered projection images in a time series taken at 3 min intervals (00:00 and 00:03, respectively). Projection images were then smoothened (average of 3 + 3 pixel neighborhood) to reduce background noise (B, B′). Then, an arbitrary threshold was applied to define the area of the cell (C, C′). The same threshold was applied to all images in a time series. D, A ‘difference image’ was generated from adjacent time-points (C and C′) using the Image Calculator ‘difference’ function in ImageJ, which calculates and displays the absolute value of differences between pixels in the two images. Thus, white areas indicate regions of change (either extension or retraction) in the cell. Note, for example, areas of change in processes (arrows). Dark regions indicate areas of little or no change in the cell, as for example in the cell soma (arrowhead). A video of this cell can be found in Supporting Information Movie 7.
GLIA_22394_sm_SuppMovie1.avi6.5 MB	Supporting Information Movie 1. Time-lapse movie of hippocampal slice during oxygen-glucose deprivation (OGD) with Sytox where GFP expressing microglia (MG) die as shown by loss of GFP followed by gain of Sytox. Length of movie: 45 min; interval between frames: 3 min.
GLIA_22394_sm_SuppMovie2.avi11.2 MB	Supporting Information Movie 2. Time-lapse movie of difference images used to quantify the motility index of microglial cells during control (left), hypoxia (middle) and OGD (right) conditions. Notice that only during control conditions are there continuously obvious changes in microglial dynamics. Length of movie: 6 hr; interval between frames: 10 min.
GLIA_22394_sm_SuppMovie3.avi6.9 MB	Supporting Information Movie 3. Time-lapse movie of a microglia (green) that is able to engulf a Sytox labeled dead cell (red) in the stratum radiatum of a hippocampus slice during OGD. Length of movie: 6 hr; interval between frames: 10 min.
GLIA_22394_sm_SuppMovie4.avi3.5 MB	Supporting Information Movie 4. Time-lapse movie of a hippocampal slice under baseline conditions (30 min), then OGD (30 min), then OGD in the presence of 200 U/mL apyrase (30 minutes). During OGD, cells in and near the stratum pyramidale (SP) retain their process motility, although cells in the stratum radiatum (SR) are inhibited. This OGD-resistant MG motility in the SP is abolished by apyrase, suggesting that it is dependent on extracellular purines. Length of movie: 90 min; interval between frames: 3 min.
GLIA_22394_sm_SuppMovie5.avi1,007.1 KB	Supporting Information Movie 5. Time-lapse movie showing raw data of several GFP expressing microglia (MG, left) and the corresponding difference images (right) during OGD in the presence of 0.5mM exogenous ATP. ATP diffuses into the slice from left to right forming a gradient. Although OGD inhibits the motility of these MG, as ATP diffuses into the slice, MG motility is restored along the gradient of ATP with more proximal MG responding earlier (within 20 min) and more distal MG responding later (within 70 minutes). Length of movie: 3 hr; interval between frames: 10 min.
GLIA_22394_sm_SuppMovie6.avi6.4 MB	Supporting Information Movie 6. Time-lapse movie from a P2 slice (left) and a P6 slice (right) during OGD labeled with Topro-3 (yellow) that, like Sytox, labels the stratum pyramidale. In each movie, two representative GFP-expressing MG are tracked (yellow and red lines) through time. Tracks of P2 MG are much more elaborate than those of P6 MG showing that OGD less effectively inhibits MG motility and migration in the P2 animal than the P6 one. Length of movie: 6 hr; interval between frames: 5 min.
GLIA_22394_sm_SuppMovie7.avi1.7 MB	Supporting Information Movie 7. Time-lapse movie including sample MG from the same slice in the stratum pyramidale (SP) (left) and stratum radiatum (SR) (right) monitored during 1 hr of baseline then 2 hr of OGD then 1 hr of washout. The SP's cell motility remains largely unchanged before, during or after OGD. Although the cell in the SR is motile during baseline, its motility is severely limited by OGD and restored after OGD. Length of movie: 4 hr; interval between frames: 3 min.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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Citing Literature

Volume60, Issue11

November 2012

Pages 1747-1760

Effects of oxygen-glucose deprivation on microglial mobility and viability in developing mouse hippocampal tissues

Abstract

INTRODUCTION