Ex vivo dynamic imaging of retinal microglia using time-lapse confocal microscopy - PubMed (original) (raw)

Ex vivo dynamic imaging of retinal microglia using time-lapse confocal microscopy

Jung Eun Lee et al. Invest Ophthalmol Vis Sci. 2008 Sep.

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.

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Figures

Figure 1

Figure 1. Distribution of microglia in the retina

(A) Agarose-embedded sections showing distribution of GFP-positive microglia (in 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 with GSIB4 lectin (red) (B) Confocal image from a retinal wholemount explant showing the distribution of ramified, GFPpositive microglia in the outer plexiform layer. (Scale bar = 50 µm) INL = inner nuclear layer, ONL = outer nuclear layer, OS = photoreceptor outer segments.

Figure 2

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 for each cellular process (indicated by arrows and numbered) demonstrate extension and retraction movements. Scale bar = 50µm. Process 7 was transient with a short half-time (not located on figure) [Supplementary online material, movie S1] (B) High-magnification confocal image of a single microglial process bearing multiple tertiary terminal processes. Image series in insets (taken 10s apart) show progressive structural changes in existing processes, de novo initiation of processes, and complete elimination of existing processes. Scale bar = 5µm. [Supplementary online material, movie S2]

Figure 3

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. [Supplementary online material, movie S3] (B) A single cell extends adjacent terminal processes whose tips meet transiently at a single point (circle). [Supplementary online material, movie S4] Scale bar = 20µm.

Figure 4

Figure 4. Distribution of structural changes over the entire microglia cell arbor

Comparison of the area occupied by a microglia cell at one point in time (A) with the area occupied by the same cell over 500s (maximum z-projection of 50 time-lapse images, captured 10s 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 time = 0s and at t = 500s shows that the extent of process additions (in green) are balanced by the extent of process retractions (in red). Scale bar = 20µm.

Figure 5

Figure 5. Morphological responses of microglia to focal laser injury

(A) Low magnification (20X) view of microglia in the vicinity of a focal laser burn (dotted circle) with colors representing depth in the Z-direction (purple=superficial and red=deep). Scale bar = 100µm. (B) Higher magnification of microglia at the edge of the laser burn (dotted circle) immediately (left) and 393s after (right) laser injury. Scale bar = 30 µm (C) Cell from inset in (B) showing progressive morphological change in response to laser injury. [Supplementary online material, movie S5] Subtraction image (right) between the initial and final images demonstrate that processes are extended in the direction of the laser burn (in green) and withdrawn on the opposite side of the cell (in red). Scale bar = 20µm. Graph of polarity coefficient vs. time after laser (lower left) demonstrates increasing polarization of the cell towards the laser burn with time. Graph of process number vs. time (lower right) shows decreasing number of primary and terminal processes after laser injury. (D) Polarity coefficients of multiple microglial cells immediately post-laser injury (0 to 5 min) and after a waiting period (30–70 minutes). Error bars indicate 95% confidence intervals. Cells were slightly but not significantly polarized <5 min after injury but became significantly polarized (p < 0.05) after 30 min. (E) Numbers of primary and terminal processes per cell are significantly reduced after 30 minutes post-laser injury (asterisks indicate p <0.05). Analysis of (D) and (E) based on 153 cells from 24 time-lapse recordings in 6 animals.

Figure 6

Figure 6. Dynamic behavior of microglia after focal laser injury

(A) Microglia cell in the vicinity of laser burn (arrow indicates location of laser burn outside image field) soon after laser injury (t = 0s) and approximately 8 minutes afterwards (t = 495s). Subtraction image (right) between these 2 time-points shows displacement of cell body position (dashed and solid circles indicate soma position at 0s and 495s respectively) in addition to a progressive extension of processes towards the laser burn. [Supplementary online material, movie S6] (B) An amoeboid microglia is seen near the laser injury site and migrates across the imaging field. [Supplementary online material, movie S8] Scale bars = 15 µm. (C) Average migration velocity of ramified microglia at different distances from the center of laser injury. The average rates of cellular migration is similar between cells up to 400 microns away from the center of laser injury, but cellular migration decreases significantly with distances greater than 400 microns from the injury site (asterisk indicates 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 to that in the resting state (n = 363 processes from 37 cells from 7 recordings in 3 animals).

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