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Figure 1.

Drawing of a spider web at each stage of the spider behavior.

(A): The spider is in the middle of the web waiting for prey to hit the web; it will sense the vibration. (B): The spider senses the fly hitting the web and begins to withdraw its legs and move in the direction of the vibration. The spider is showing directional orientation toward the fly. (C): The spider has left the center of the web and is moving along the primary tension member of the web in the direction of the fly. (D): The spider comes in contact with the fly, seizes the fly, wraps it or eats it. (E): When the fly is consumed the spider starts the return to the center of the web. Again, the orientation of the spider is away from the original disturbance and toward the center of the web. (F): The spider, back at the center of the web, has re-extended its legs to the primary tension members of the web waiting until for another disturbance, or a new meal. The direction of the action is driven by a vibration disturbance that is sensed in the legs of the spider. Microglia appear to use their processes as spider legs and move to the injured site.

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Figure 2.

Drawing of the olfactory bulb (A) and the location of the injury and the sampling area. (B): Photomicrograph of the sagittal section of olfactory bulb. The sample grid used for every animal includes both the yellow and red areas and were further broken down into Locations I, II, and III. This was to show the movement and depletion of the microglia from these areas and their subsequent return. Sample grid: 150 µm.

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Figure 3.

Photographs of microglia cells in the olfactory bulb 2 days after a standardized transection of the olfactory bulb sectioned and stained with Iba-1.

These photomicrographs are showing the cells advancing. (A and B): Stage 1A, a microglia cell has processes that are ramified and spread out, with a small soma. (C–D): Stage 2A, the soma has increased in size to approximately 1.5–2 times the soma diameter of a stage 1A cell. The cell processes have started to retreat and the branches next to the cell soma are thickened. (E–F): Stage 3A, the cell soma diameter is enlarged to 2–3 times the soma diameter of stage 1A. All cell processes have retracted and thickened. (G–H): Stage 4A, the soma diameter is 3–4 times larger than the stage 1 soma; all thin cell processes have completely retracted and only the thick cell branches remain. (I–J): Stage 5A, soma diameter is 5 times larger than the soma diameter in stage 1; the thick process is replaced by a thin process oriented in the direction of the cell movement. All branches are gone. (K–L): Stage 6A shows the transformation from microglia to macrophage. The microglia cell has a large round morphology with a large soma, with one short or no processes. (Scale bar: 10 µm).

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Figure 4.

Relative microglial cell density in regions at zero µm (I), 200 µm (II) and 400 µm (III) from the injury site at 2, 7 and 14 days after experimental transection of the olfactory bulb, stratified into 6 stages according to the morphology of resting versus activated cells.

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Figure 5.

The drawing are tracings of the resting activated microglia starting in resting stage 1A, continue to an activated stage 6A (macrophage stage), transform to stage 6R (multinuclear cells), and then return to a resting stage 1R.

The blue arrows indicate the increase in activation and the green arrow indicate the transition from activated to returning. This demarcation appears to be the point that the macrophages consume other cells and debris and are digesting or carrying it away from the event horizon. Note that the cells of stage 6R to stage 3R appear larger than the cells of other stages. This is possibly due to the increased number of nuclei that they have consumed. Some of these nuclei and cell debris appeared to have been transferred to other macrophages.

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Table 1.

List of characteristics of each stage of microglial activation and de-activation.

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Figure 6.

Photographs of microglia cells in the olfactory bulb 7 days after a standardized transection of the olfactory bulb sectioned and stained with IBA-1 (green).

(A): Stage 2R, the soma decreased in size to about 1.5–2 times the soma diameter of a stage 1A cell; the cell processes lengthened and the cell lost its directional orientation. Branches were clearly prominent on the processes. (B): Stage 1R, the microglia cell had a small soma with processes that were ramified and spread. The cell was back to its original location and separated from the other neighboring microglia (Scale bars: 10 µm).

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Figure 7.

Photomicrographs of sagittal sections of the olfactory bulb with the experimental incision injury site to the right.

(A): Day 2 post injury. Note the accumulation of small round cells at the injury site (at the right edge of photo); the orientation of the cell was in direction of the injury site. (B): Day 7 post injury. The small round cell bodies partially disappeared and the cell orientation was away from the injury site. The cells appeared to be returning to the tissue. (C): Day 14 post injury. There were only a few areas that still had high concentrations of round microglia cell bodies/macrophages; the cells were oriented away from the injury site and some highly branched cells were present distant to the injury site. The green was Iba-1 with an Alexa dye.

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Figure 8.

Microglial cell density (cells/mm2) at 2, 7 and 14 days after experimental transection of the olfactory bulb in regions at zero µm, 200 µm and 400 µm from the injury site.

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Figure 9.

Graph showing the distribution of microglial cell processes oriented toward (green bars, UP) or away (purple bars, Down) from an experimental transection site of the olfactory bulb the cut at 2, 7, and 14 days after the injury.

The blue bar is the net direction of the processes.

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Figure 10.

Graph showing the mean number of processes per microglial cell at zero µm (area I), 200 µm (area II) and 400 µm (area III) from the injury site at 2, 7 and 14 days after experimental transection of the olfactory bulb.

Control animal had an average of 5.8 processes per cell. The bars show the standard deviation.

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Figure 11.

Multiphoton Z-Stack images of microglia cells immunohistochemically stained by Iba-1 to show the cell bodies and cell processes.

The panels show a Z-Stack of images while the panels to the right and top demonstrate the level of the image in the stack. (A): partially activated microglia with a few activated cells. The cross hairs show the location of the cell and the upper and right panels show the depth of the cell. (B): activated microglial cells as by increased size and more circular shape size. (C): fully activated microglia with several microglia cells in stage 5R. Note: the cells cover the full thickness of the histological slide and although some cells are located close to each other, they can clearly delineated from each other by the Iba-1 label in the cell walls. Scale bar: 10 µm.

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