September 2008
Volume 49, Issue 9
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Retinal Cell Biology  |   September 2008
Ex Vivo Dynamic Imaging of Retinal Microglia Using Time-Lapse Confocal Microscopy
Author Affiliations
  • Jung Eun Lee
    From the Office of the Scientific Director, the
    Division of Epidemiology and Clinical Research, and the
  • Katharine J. Liang
    From the Office of the Scientific Director, the
    Division of Epidemiology and Clinical Research, and the
  • Robert N. Fariss
    Biological Imaging Core, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
  • Wai T. Wong
    From the Office of the Scientific Director, the
Investigative Ophthalmology & Visual Science September 2008, Vol.49, 4169-4176. doi:10.1167/iovs.08-2076
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      Jung Eun Lee, Katharine J. Liang, Robert N. Fariss, Wai T. Wong; Ex Vivo Dynamic Imaging of Retinal Microglia Using Time-Lapse Confocal Microscopy. Invest. Ophthalmol. Vis. Sci. 2008;49(9):4169-4176. doi: 10.1167/iovs.08-2076.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Retinal microglia have been implicated in the pathogenesis of various retinal diseases, but their basic function and cellular phenotype remain incompletely understood. Here, the authors used a novel ex vivo retinal imaging preparation to examine the behavioral phenotype of living retinal microglia in intact tissue and in response to injury.

methods. Fluorescence-labeled microglia in retinal explants from CX3CR1+/GFP transgenic mice were observed using time-lapse confocal imaging. High spatial and temporal resolution imaging parameters were used to follow dynamic microglial behavior in real time.

results. Under normal conditions, resting retinal microglia are not static in structure but instead exhibit extensive structural dynamism in their cellular processes. Process movements are highly random in direction but are balanced to maintain overall cellular symmetry and arbor size. At rest, however, these exuberant process movements do not result in overt cellular migration. After focal laser injury, microglial processes increase significantly in their motility and direct themselves toward the injury site. Microglia rapidly transition their morphologies from symmetric to polarized toward the laser lesion. Microglia also transition from a fixed to a migratory phenotype, translocating through tissue while retaining their ramified morphology.

conclusions. Retinal microglia normally occupying uninjured tissue display a continuous, dynamic behavior that suggests functions of tissue surveillance and intercellular communication. Microglial behavior is highly regulated by, and immediately responsive to, focal tissue injury and may constitute a therapeutic cellular response to focal laser photocoagulation. Ex vivo live imaging in the retina is an experimental approach well suited to the study of dynamic aspects of microglial physiology.

Microglial cells are resident immune cells of the central nervous system. 1 2 Akin to macrophages, microglia play a key role in innate and adaptive immunity, mediating immune tissue protection against infection and neuronal insult. 3 However, they can also be involved in exacerbating tissue damage through neurotoxic and proinflammatory effects when fully activated. 4 Unlike brain microglia, which have a broad distribution throughout neural tissue, retinal microglia exhibit a stratified distribution correlating to the laminar organization of the retina. 5 In normal adult retina, microglia are restricted to the inner retinal layers and are largely absent from the region extending from the outer nuclear layer to Bruch membrane. 6  
Similar to their counterparts in the brain, retinal microglia have also been implicated in a number of retinal degenerative, inflammatory, and vascular diseases. 7 8 9 Activated retinal microglia have been found in human tissue histopathologic specimens of retinal disease where pathologic changes are found. 10 11 12 Abnormal accumulation and altered morphologies of retinal microglia have also been found in animal models of retinal disease, including retinal detachment, 9 retinitis pigmentosa 8 13 age-related macular degeneration, 14 15 and diabetic retinopathy. 16 In the latter, studies in mouse models 16 and human disease 12 demonstrate that morphologic changes in retinal microglia occur early in disease progression, suggesting that these may have a inductive role in causing pathologic neuronal and vascular changes. Numerous studies have demonstrated that microglia, in general, display a multifaceted array of structural phenotypes that can change markedly, depending on the tissue context. 17 18 These phenotype changes include alterations in cellular morphology, tissue distribution, migratory characteristics, and changes in process structure. These structural phenotypes are thought to reflect different functional modes, enabling microglia to effect different changes in the surrounding tissue. 2 In the retina, characterizations of these microglial phenotypes under normal and disease conditions, and how they reflect on changing microglia function, are not well understood. In addition, how various therapeutic interventions in retinal disorders may cause changes in microglia physiology is unknown. 
In this study, we used a novel ex vivo imaging approach to study not only the structure but also the behavior of living retinal microglia in intact tissue. Previous studies have shown that microglia in the cerebral cortex demonstrate structural dynamism in their ramified processes, 19 20 but whether this behavior extends to areas outside the brain, specifically to the retina, has not been examined. Although recent studies have examined how microglia may accumulate and traffic in the retina in vivo over hours to days, 21 the higher spatial and temporal resolution of our imaging system enabled us to follow detailed changes in the structure of individual microglial processes and to quantitate process and migration velocities. In addition, our system was amenable to examining changes in microglia behavior under different conditions. Here, we examined changes in microglial behavior before and after focal laser treatment using parameters similar to those used in the grid laser treatment of diabetic retinopathy. Our results provide a description of baseline microglia behavior at rest and of how this behavior is regulated by focal injury. Taken together, our findings provide a perspective on the functional relevance of changing retinal microglia behavior in general and raise hypotheses about the mechanism underlying focal laser treatment for diabetic macular edema in particular. 
Materials and Methods
Experimental Animals
CX3CR1GFP/GFP mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). Heterozygous CX3CR1+/GFP were created by crossing CX3CR1GFP/GFP mice to wild-type C57BL/6 mice and housed and bred in National Institutes of Health (NIH) animal facilities. Experiments were conducted according to protocols approved by a local Institutional Animal Care and Use committee. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Vibratome Sectioning and Immunohistochemistry
Retinal tissue, fixed in 4% paraformaldehyde in 1× PBS, pH 7.3, was embedded in 7% agarose and sectioned with a vibrating microtome (Leica; VT1000S, Bannockburn, IL). Immunohistochemical staining was performed with Griffonia simplicifolia IB4 (GSIB4) lectin, conjugated with Alexa 568 (1:100) to reveal vascular staining, 4′,6-diamidino-2-phenylindole (DAPI) (1:1000) to act as a nuclear marker (Invitrogen, Carlsbad, CA), and Iba-1 (1:500) to label microglia (Wako Chemicals, Richmond, VA). 
Retinal Explant Wholemount Preparation
Mice in groups ranging from neonatal to 5 weeks of age were humanely killed, and their eyes were enucleated. Eyecups were immediately immersed in ice-cold oxygenated Ringer solution (125 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 0.75 mM MgCl2/6 H2O, 1.25 mM NaH2PO4, 10 mM d-glucose, 20 mM HEPES, pH 7.35–7.45). Retinas were dissected from the eyecups and then mounted on black Millipore filter paper (HABP045; Millipore, Billerica, MA) with the ganglion cell layer uppermost, as described previously. 22  
Time-Lapse Confocal Microscopy
Wholemount retina preparations were transferred to a stage-mounted, temperature-controlled (32°C) chamber (Bioptechs, Butler, PA) through which oxygenated Ringer solution was continuously superfused (30–60 mL/h). Tissue preparations were imaged using a confocal microscope (SP2; Leica, Exton, PA) and a 40× (0.80 numerical aperture) or a 63× (0.90 numerical aperture) water-immersion objective. Multiplane Z series time-lapse images were collected at a 1024 × 1024 or 512 × 512 pixel resolution. Image stacks traversing volumes of interest were captured every 10 seconds. 
Argon Laser Injury
Retinal tissue mounted on filter paper supports were placed perpendicularly to the light path of a slit lamp–mounted 532-nm photocoagulation laser (Iridex, Mountain View, CA). The aiming beam was focused on the plane of the retinal explant, and two to three isolated focal laser burns were delivered. Laser settings used were as follows: power, 110 mW; duration, 100 ms; spot size, 50 μm. Initial images were captured approximately 3 minutes after focal laser injury. 
Image Processing
Image processing was performed using NIH ImageJ software. Two-dimensional (2D) representations of 3D microglial structures were created from maximum intensity projections in the z-dimension, and recursively aligned in the time dimension, and time-lapse movies were created. Quantitative analysis of microglia processes were limited to those fully contained in the imaging space, avoiding focus change artifacts that might have been mistaken for structural changes. Mean microglial process velocities were calculated by tracking the ends of terminal processes over time using the new ImageJ particle tracker, Plugin MTrackJ, and quantitating the average rate of change of process displacement over the duration of the recording. Pixel-counting algorithms were used to quantify the 2D area occupied by each cell at a single time point and across multiple time points from binarized z-projections of entire cellular arbors. Mean microglial migration velocities were calculated by tracking the centers of microglia soma over time and quantitating the average rate of soma displacement over the duration of the recording. Directional migration velocity was calculated from the change in distance of the cell soma relative to the center of the laser lesion (with a negative value representing movement toward the site injury) and dividing by the time interval of observation. 
To characterize the polarization of cellular processes toward the site of laser injury for each individual cell, distances of all terminal processes were measured from the center of laser injury. A polarization coefficient, P, was defined as  
\[P{=}(\overline{D_{\mathrm{p}}{-}D_{\mathrm{s}}})/K_{\mathrm{d}}\]
where D p = distance of process from center of laser injury, D s = distance of soma from center of laser injury, and K d = average diameter of microglia cells. A P of 1 represents a physical extreme of polarization where the tips of all cellular processes were located on one end of an arbor and the soma were located on the other end, whereas a P of 0 represents a cell with the ends of terminal processes equally distributed between the half of the arbor closer to the injury and the other half further away. Polarization toward the laser injury is expressed as a negative value by this convention. 
Statistical Analysis
Results were analyzed by two-tailed Student’s t-test and are expressed as mean ± SEM (unless otherwise indicated). 
Results
Distribution of Microglia in the Retina
We examined the distribution of green fluorescence protein (GFP)–labeled cells in the retina of young adult CX3CR1+/GFP mice between 4 and 5 weeks of age. Previous immunohistochemical studies of GFP-labeled cells in the cerebral cortex 23 and retina 14 of these transgenic animals have identified the cells as microglia based on the expression of cell-specific markers. Similarly, we verified that all GFP-positive cells in the retina were also positive for Iba-1, a marker for microglia in the central nervous system (CNS; data not shown). GFP-labeled microglia in CX3CR1+/GFPmice had a distribution similar to that found in wild-type mice. 6 In retinal cross-sections, most microglia were located in the inner half of the retina and exhibited a horizontal laminar distribution extending from the nerve fiber layer to the outer plexiform layer 1 , with only an occasional cell in the outer nuclear layer or photoreceptor outer segments. Examination of GFP-labeled microglia in retinal flat mounts from CX3CR1+/GFP mice also revealed a regular distribution of microglia, with uniform spacing between adjacent cell soma of 73.1 ± 2.1 μm (n = 55 cells in 5 animals) and ramified processes extending over territories (which we refer to as arbors) that are spaced regularly and show little overlap 1
Dynamic Behavior of Resting Retinal Microglia
To study the behavior of retinal microglia under uninjured conditions (the so-called resting microglia), we isolated living flat-mount retina explants from adult CX3CR1+/GFPmice. Acutely isolated explants were maintained in a temperature-controlled chamber through which oxygenated medium was perfused. Microglia in retinal explants had a typical resting morphology in which primary processes emerging from the cell body branch into smaller terminal processes of variable lengths and shapes, often ending in bulbous tips 2 . Time-lapse confocal microscopy was used to follow changes in structure and morphology of GFP-positive microglia under physiological conditions. We found that the processes of retinal microglia in the resting state are not static but are highly dynamic, changing their structure at remarkably rapid rates (Movie S1; all Movies are online at http://www.iovs.org/cgi/content/full/49/9/4169/DC1). Primary and terminal processes showed sustained changes in shape and length that occurred in seconds. In a single cell, these changes consisted of simultaneous extensions and retractions of existing processes, de novo formation of new processes, and process elimination (2 ; Movie S2). 
Under resting conditions, process movements did not appear to be directed toward any particular orientation; processes extended in all directions, appearing to sample the surrounding extracellular space in a random fashion. Although the individual cell arbors were sometimes asymmetric, there was no overall polarization in any one particular direction. The rapid process movements also did not result in significant changes to the overall area of the cell arbor over the duration of recordings. Despite dynamic behavior on the level of microglial processes, the positions of cell bodies were relatively fixed, maintaining their spacing between neighboring cells. 
Although the motile processes extend and retract rapidly in an apparently random and repeated fashion, processes of a single cell generally do not cross over each other’s territories, a phenomenon that holds true for processes of the same cell. However, on occasion, the ends of terminal processes of adjacent cells can be seen extending to a common point to make transient contact before retracting (3 ; Movie S3). In some cases, the ends of terminal processes from the same cell can also be seen making transient contact (3 ; Movie S4). 
We also examined resting microglial behavior in developing retina from animals aged postnatal day (P)0 through P21. Developing microglia exhibited similar patterns of process motility (data not shown), indicating that dynamic microglia behavior is a property of developing and mature systems. 
Quantification of Resting Microglial Dynamics
To quantitate the rate of process movement, we measured for each process the rate of change of displacement of its terminal end. The mean velocity of microglial processes of resting microglia was 5.44 ± 2.33 μm/min (mean ± SD; n = 367 processes from 37 cells in 3 animals). These extremely rapid process movements have the effect of allowing the cell to sample the physical space in its “territory” repeatedly over a short period and thus to occupy a larger cellular volume over time than it occupies at any single point in time 4 4 . In 2D maximum-intensity projections of time-lapse recordings, we computed a coverage factor that compared the total area occupied by a cellular arbor over a recording period of 8 minutes with that occupied at a single time point. This coverage factor was calculated to be approximately 315% ± 14.4% (n = 89 cells from 4 animals), indicating at least a threefold increase in the extent of space-filling achieved over 8 minutes when comparing a dynamic cell to an otherwise static cell. Despite these rapid changes, the overall sum of process additions and subtractions was balanced in all cells examined (n = 14 cells from 5 animals), with maintenance of overall size of the cell territory over time 4
Morphologic Response of Retinal Microglia to Focal Laser Injury
In addition to characterizing dynamic microglia behavior in the retina under resting conditions, we also examined how microglia behavior changes in response to focal retinal injury. Focal photocoagulative injury was administered to CX3CR1+/GFP retinal explants using an argon laser on a slit lamp platform. The diameter of a typical laser burn measured approximately 50 to 100 μm, similar to that of laser photocoagulation delivered in the treatment of diabetic macular edema 5 . Higher magnification imaging showed that within 5 minutes after laser burn, microglia in the immediate vicinity (<500 μm) of the laser spot directed processes preferentially toward the injury site. This tropism of processes toward the center of injury became more marked with increasing time after injury. Microglia progressively extended new processes toward the laser lesion while withdrawing processes on the side of the cell furthest from the injury (5 5 ; Movie S5). 
To reflect the directed polarity of cellular morphologies, we computed a polarity coefficient that takes into account the distribution of cellular processes on either side of the cell with respect to the location of focal injury. 5illustrates an example of a microglia cell with increasing polarization of its structure after laser injury. The increased polarization of this cell over time is qualitatively apparent by its changing morphology and can be quantified by an increasingly negative polarity coefficient (5 , bottom left). 
We observed that, in addition to increasing polarity, postinjury microglial morphologies also became progressively less branched, with an overall decrease in primary and terminal processes (5 , bottom right). As time progressed, most microglia within a 500-μm radius of the injury extended fewer but longer processes toward the injury site. Microglia generally became significantly polarized 30 minutes or more after injury 5 , with fewer branched structures 5 . The extent of polarization, however, did not vary significantly with distance from the center of injury within the 500-μm radius (data not shown). 
Dynamic Response of Retinal Microglia Processes to Focal Laser Injury
Unlike resting microglia, which maintained stationary cell body positions despite dynamic process motility, post-laser injury microglia acquired a migratory capacity, as demonstrated by the detectable displacement of their cell somata during a recording. These cellular migrations were significantly slower than the rate of process movements, averaging 0.45 ± 0.48 μm/min (mean ± SD; n = 150 cells from 11 recordings in 5 animals; 6 ). Not all microglia were migratory after laser injury, and the rate of migration was also variable between cells (Movies S6, S7). These migratory microglia retained their highly ramified morphologies and were capable of restructuring their processes and translocating through tissue at the same time. Occasionally, microglia with activated, amoeboid morphologies were seen migrating at a higher than average rate (6 ; Movie S8). The movements of these amoeboid cells were not included in the calculation of average migration velocity. Migration velocity varied with soma-to-laser injury distance, decreasing significantly (P < 0.02) for cells outside a 400-μm radius of the lesion 6 . Although migratory movements in some cells were in the direction of the laser lesion, computations of migrational directionality on the whole did not reveal a consistent tropism toward or away from the site of injury over the relatively short time scale of the recordings (data not shown). 
Quantitation of process motility also revealed that average process velocity was markedly elevated after laser injury, measuring 9.06 ± 3.37 μm/min (mean ± SD; n = 363 processes from 99 cells in 3 animals; 6 ) or approximately 167% of that in resting microglia. The effect of increased motility also translated to increased microglial coverage of the surrounding intercellular space; maximum intensity projections over 8 minutes of recording showed a coverage factor of 384% ± 12%, significantly higher than the 315% calculated for resting microglia. 
Discussion
We have used an intact retinal explant ex vivo system to examine the nature of retinal microglial process dynamics. Unlike acute brain slice preparations in which the slicing procedure results in extensive tissue damage close to resident microglia, retinal wholemount preparations isolate retinal tissue as an intact sheet and preserve neuronal circuitry and retinal vasculature, thus maintaining neuronal connectivity and cellular behavior, as demonstrated by other experiments making use of this preparation. 24 25 Retinal microglia in wholemount preparations were observed to retain their ramified morphology and laminar distribution many hours after acute isolation and did not progress through morphologic changes typical of microglia activation seen in brain slice preparations. 26 GFP-labeled retinal microglia in similar transgenic mice have been imaged in vivo using confocal scanning laser ophthalmoscopy in which microglia distribution and migration were monitored after laser injury over hours to days. 21 27 Individual cellular morphologies and processes could not, however, be visualized and followed. Our imaging system affords a higher spatial and temporal resolution that complements those observations by visualizing microglial behavior continuously on the level of the individual cell and the individual process. 
Our results show that so-called resting retinal microglia show a rapid dynamism in their processes that is continuous and sustained but is unaccompanied by cellular migration on the level of the soma. These process movements appear random, involve primary and terminal branches of the cellular arbor, and occur in all directions. Extrapolation of process movements over time reveal that resting retinal microglia are capable of sampling their entire extracellular spaces repeatedly over a span of minutes. These observations support an endogenous surveillance function for rapid process motility in resting microglia. Our observations of transient, coordinated contact between terminal microglial processes among otherwise random patterns of motility suggest in addition that process dynamics may also serve to exchange signals between neighboring microglia. These communications may explain why retinal microglia distribution in the neural parenchyma occurs in a laminar, tiled fashion with little overlap between arbors, and they explain how seemingly random movements are limited to each cellular arbor’s territory. Taken together, our observations indicate that rapid and continuous microglial coverage of the inner retina is a constitutive and constant physiological property of the intact and healthy retina. 
Our observations of the behavior of resting retinal microglia resemble those documented for microglia in the cerebral cortex 19 20 and other CX3CR1-positive immune cells in visceral organs 28 29 in terms of pattern and nature. The average velocity of moving microglial process was considerably higher in the retina than the cerebral cortex (5.4 ± 2.3 μm/min vs. 1.5 ± 0.1 μm/min). 20 Although the two systems are clearly different (ex vivo vs. in vivo retinal explant in an anesthetized animal), further investigation of the effects of neuronal activity or metabolic rate on microglial dynamics may provide insight for the evaluation of resting microglia behavior across specialized areas of the CNS. 
The function of retinal microglia process dynamics may also be illuminated by an evaluation of microglial response to focal injury. We found that process dynamics were altered after injury in a number of significant ways. First, average process velocity near the laser burn increased significantly by 67% in the first hour after laser injury. Second, the process movements changed from a state without orientation to one clearly directed toward the laser lesion. Third, the overall balance between process additions and eliminations was shifted to result in a polarized and simplified cellular arbor. Fourth, some retinal microglia acquired a post-injury migratory phenotype that allowed translocation through the retina while maintaining rapid process motility. The migratory behavior of polarized retinal microglia was not demonstrably directed toward the laser lesion, though there may be a net accumulation of microglia in the vicinity of the laser lesion over longer periods of time. 21 The migration exhibited by ramified, polarized microglia appeared to be distinct from the rapid locomotion of activated, amoeboid microglia lacking a ramified morphology. The latter were seen infrequently after laser injury in this preparation whereas they can be widely found in brain slice preparations. 26 These microglia in postlaser retina probably represent a smaller subset of reactive microglia that have been activated to a greater degree and have transitioned to an amoeboid, highly migratory morphology. This phenotypic diversity in retinal microglia after injury reflects a similar diversity of potential microglial responses that are likely to vary with injury intensity and dose and that may underlie different aspects of tissue response. 2  
The directed and distance-dependent nature of microglial response to laser injury suggests a role for diffusible chemoattractant signals originating from the injury site that upregulate process motility, reorient microglial morphology, and confer migratory behavior. Other studies have indicated that extracellular ATP, acting through P2Y receptors, may be involved in microglial response to injury. 19 30 In this study we have used laser injury parameters that simulate retinal laser burns delivered in the treatment of diabetic macular edema. We speculate that the changing behavior of retinal microglia from a resting, stationary state to a polarized, migratory state with increasingly rapid surveying movements may underlie tissue responses after focal laser treatment in retinal disease. Retinal microglia may exist in a variety of activity states 2 and may shift from a resting to an alerted state after local injury. Alerted microglia may release neurotrophic factors and inflammatory mediators that exert beneficial vasculoprotective and neuroprotective effects that underlie the therapeutic benefit seen in focal laser treatment for diabetic macular edema. 31 Future investigations of factors that regulate microglial behavior in normal retina and in models of retinal disease may further clarify what endogenous roles retinal microglia play under normal conditions and how they may contribute to the pathogenesis of retinal disorders. 
Figure 1.
 
Distribution of microglia in the retina. (A) Agarose-embedded sections showing distribution of GFP-positive microglia (green) in CX3CR1+/GFP animals in the inner retina, primarily in the ganglion cell layer (GCL), inner plexiform layer (IPL), and outer plexiform layer (OPL). Nuclei were labeled with DAPI (blue), and vessels were labeled with GSIB4 lectin (red). (B) Confocal image from a retinal wholemount explant showing the distribution of ramified, GFP-positive microglia in the outer plexiform layer. Scale bar, 50 μm. INL, inner nuclear layer; ONL, outer nuclear layer; OS, photoreceptor outer segments.
Figure 1.
 
Distribution of microglia in the retina. (A) Agarose-embedded sections showing distribution of GFP-positive microglia (green) in CX3CR1+/GFP animals in the inner retina, primarily in the ganglion cell layer (GCL), inner plexiform layer (IPL), and outer plexiform layer (OPL). Nuclei were labeled with DAPI (blue), and vessels were labeled with GSIB4 lectin (red). (B) Confocal image from a retinal wholemount explant showing the distribution of ramified, GFP-positive microglia in the outer plexiform layer. Scale bar, 50 μm. INL, inner nuclear layer; ONL, outer nuclear layer; OS, photoreceptor outer segments.
Figure 2.
 
Resting retinal microglia show marked process motility. (A) Microglial cell in a CX3CR1+/GFP mouse retina imaged with time-lapse confocal microscopy. Length-versus-time profiles in micrometers for each cellular process (indicated by arrows and numbered) demonstrate extension and retraction movements. Scale bar, 50 μm. Process 7 was transient and had a short half-time (not located on figure; Movie S1). (B) High-magnification confocal image of a single microglial process bearing multiple tertiary terminal processes. Image series in insets (taken 10 seconds apart) show progressive structural changes in existing processes, de novo initiation of processes, and complete elimination of existing processes (Movie S2). Scale bar, 5 μm.
Figure 2.
 
Resting retinal microglia show marked process motility. (A) Microglial cell in a CX3CR1+/GFP mouse retina imaged with time-lapse confocal microscopy. Length-versus-time profiles in micrometers for each cellular process (indicated by arrows and numbered) demonstrate extension and retraction movements. Scale bar, 50 μm. Process 7 was transient and had a short half-time (not located on figure; Movie S1). (B) High-magnification confocal image of a single microglial process bearing multiple tertiary terminal processes. Image series in insets (taken 10 seconds apart) show progressive structural changes in existing processes, de novo initiation of processes, and complete elimination of existing processes (Movie S2). Scale bar, 5 μm.
Figure 3.
 
Terminal ends of microglial processes make occasional transient contact with each other. (A) Neighboring microglia progressively extend their processes to meet transiently at a common point (circle) before disengaging and retracting (Movie S3). (B) A single cell extends adjacent terminal processes whose tips meet transiently at a single point (circle; Movie S4). Scale bar, 20 μm.
Figure 3.
 
Terminal ends of microglial processes make occasional transient contact with each other. (A) Neighboring microglia progressively extend their processes to meet transiently at a common point (circle) before disengaging and retracting (Movie S3). (B) A single cell extends adjacent terminal processes whose tips meet transiently at a single point (circle; Movie S4). Scale bar, 20 μm.
Figure 4.
 
Distribution of structural changes over the entire microglia cell arbor. Comparison of the area occupied by a microglia cell at one point (A) with the area occupied by the same cell over 500 seconds (maximum z-projection of 50 time-lapse images captured 10 seconds apart) (B) demonstrates the ability of dynamic cellular processes to sample a large volume of extracellular space. (C) Subtraction image between confocal images captured at 0 second and at t = 500 seconds shows that the extent of process additions (green) are balanced by the extent of process retractions (red). Scale bar, 20 μm.
Figure 4.
 
Distribution of structural changes over the entire microglia cell arbor. Comparison of the area occupied by a microglia cell at one point (A) with the area occupied by the same cell over 500 seconds (maximum z-projection of 50 time-lapse images captured 10 seconds apart) (B) demonstrates the ability of dynamic cellular processes to sample a large volume of extracellular space. (C) Subtraction image between confocal images captured at 0 second and at t = 500 seconds shows that the extent of process additions (green) are balanced by the extent of process retractions (red). Scale bar, 20 μm.
Figure 5.
 
Morphologic responses of microglia to focal laser injury. (A) Low-magnification (20×) view of microglia in the vicinity of a focal laser burn (dotted circle) with colors representing depth in the z-direction (purple, superficial; red, deep). Scale bar, 100 μm. (B) Higher magnification of microglia at the edge of the laser burn (dotted circle) immediately (left) and 393 seconds after (right) laser injury. Scale bar, 30 μm. (C) Cell (B, inset) showing progressive morphologic change in response to laser injury (Movie S5). Subtraction image (right) between the initial and final images demonstrate that processes are extended in the direction of the laser burn (green) and are withdrawn on the opposite side of the cell (red). Scale bar, 20 μm. Graph of polarity coefficient versus time after laser (lower left) demonstrates increasing polarization of the cell toward the laser burn with time. Graph of process number versus time (lower right) shows decreasing number of primary and terminal processes after laser injury. (D) Polarity coefficients of multiple microglial cells immediately after laser injury (0–5 minutes) and after a waiting period (30–70 minutes). Error bars indicate 95% confidence intervals. Cells were slightly but not significantly polarized less than 5 minutes after injury but became significantly polarized (*P < 0.05) after 30 minutes. (E) Numbers of primary and terminal processes per cell are significantly reduced 30 minutes after laser injury (*P < 0.05). Analysis of (D) and (E) based on 153 cells from 24 time-lapse recordings in six animals.
Figure 5.
 
Morphologic responses of microglia to focal laser injury. (A) Low-magnification (20×) view of microglia in the vicinity of a focal laser burn (dotted circle) with colors representing depth in the z-direction (purple, superficial; red, deep). Scale bar, 100 μm. (B) Higher magnification of microglia at the edge of the laser burn (dotted circle) immediately (left) and 393 seconds after (right) laser injury. Scale bar, 30 μm. (C) Cell (B, inset) showing progressive morphologic change in response to laser injury (Movie S5). Subtraction image (right) between the initial and final images demonstrate that processes are extended in the direction of the laser burn (green) and are withdrawn on the opposite side of the cell (red). Scale bar, 20 μm. Graph of polarity coefficient versus time after laser (lower left) demonstrates increasing polarization of the cell toward the laser burn with time. Graph of process number versus time (lower right) shows decreasing number of primary and terminal processes after laser injury. (D) Polarity coefficients of multiple microglial cells immediately after laser injury (0–5 minutes) and after a waiting period (30–70 minutes). Error bars indicate 95% confidence intervals. Cells were slightly but not significantly polarized less than 5 minutes after injury but became significantly polarized (*P < 0.05) after 30 minutes. (E) Numbers of primary and terminal processes per cell are significantly reduced 30 minutes after laser injury (*P < 0.05). Analysis of (D) and (E) based on 153 cells from 24 time-lapse recordings in six animals.
Figure 6.
 
Dynamic behavior of microglia after focal laser injury. (A) Microglia cell in the vicinity of laser burn (arrow, location of laser burn outside image field) soon after laser injury (t = 0 seconds) and approximately 8 minutes afterward (t = 495 seconds). Subtraction image (right) between these two times shows displacement of cell body position (dashed and solid circles indicate soma position at 0 seconds and 495 seconds, respectively) in addition to a progressive extension of processes toward the laser burn (Movie S6). (B) An amoeboid microglia is seen near the laser injury site and migrates across the imaging field (Movie S8). Scale bars, 15 μm. (C) Average migration velocity of ramified microglia at different distances from the center of laser injury. Average rates of cellular migration were similar between cells up to 400 μm away from the center of laser injury, but cellular migration decreased significantly with distances greater than 400 μm from the injury site (*P < 0.05). (D) Average velocity of microglia processes after laser injury (n = 363 processes from 99 cells from 12 recordings in 3 animals) increased significantly (*P < 0.05) compared with that in the resting state (n = 363 processes from 37 cells from 7 recordings in 3 animals).
Figure 6.
 
Dynamic behavior of microglia after focal laser injury. (A) Microglia cell in the vicinity of laser burn (arrow, location of laser burn outside image field) soon after laser injury (t = 0 seconds) and approximately 8 minutes afterward (t = 495 seconds). Subtraction image (right) between these two times shows displacement of cell body position (dashed and solid circles indicate soma position at 0 seconds and 495 seconds, respectively) in addition to a progressive extension of processes toward the laser burn (Movie S6). (B) An amoeboid microglia is seen near the laser injury site and migrates across the imaging field (Movie S8). Scale bars, 15 μm. (C) Average migration velocity of ramified microglia at different distances from the center of laser injury. Average rates of cellular migration were similar between cells up to 400 μm away from the center of laser injury, but cellular migration decreased significantly with distances greater than 400 μm from the injury site (*P < 0.05). (D) Average velocity of microglia processes after laser injury (n = 363 processes from 99 cells from 12 recordings in 3 animals) increased significantly (*P < 0.05) compared with that in the resting state (n = 363 processes from 37 cells from 7 recordings in 3 animals).
 
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