The Spider Effect: Morphological and Orienting Classification of Microglia in Response to Stimuli in Vivo

The different morphological stages of microglial activation have not yet been described in detail. We transected the olfactory bulb of rats and examined the activation of the microglial system histologically. Six stages of bidirectional microglial activation (A) and deactivation (R) were observed: from stage 1A to 6A, the cell body size increased, the cell process number decreased, and the cell processes retracted and thickened, orienting toward the direction of the injury site; until stage 6A, when all processes disappeared. In contrast, in deactivation stages 6R to 1R, the microglia returned to the original site exhibiting a stepwise retransformation to the original morphology. Thin highly branched processes re-formed in stage 1R, similar to those in stage 1A. This reverse transformation mirrored the forward transformation except in stages 6R to 1R: cells showed multiple nuclei which were slowly absorbed. Our findings support a morphologically defined stepwise activation and deactivation of microglia cells

Factorial analysis of adaptable properties of self-assembling peptide matrix on cellular proliferation and neuronal differentiation of pluripotent embryonic carcinoma

An integrative and quantitative approach for systematically studying the effects of changing the matrix environment on pluripotent cell viability and neuronal differentiation was demonstrated. This approach, based on factorial analysis and a self-assembling peptide (SAP) matrix, was exemplified using P19 as a pluripotent cell model. In a two-level, three-factor factorial design of experiments, three niche factors, namely, culture dimensionality, fixed biochemical signal and mechanical stiffness, were simultaneously investigated. We found that cell growth was slowed in matrices containing IKVAV epitopes on the SAP constructs, and neuronal differentiation was promoted synergistically by culturing in a three-dimensional matrix and in the presence of IKVAV. Variation of the storage modulus from around 262 Pa to 672 Pa had no significant effect on either viability or differentiation. This approach should be applicable to studying how niche properties that are tunable using SAPs affect the behavior of pluripotent cells in general, thus generating guidelines for constructing artificial matrices

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.

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

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.

<p>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.</p

Figure 2

<p>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.</p

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.

<p>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.</p

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

<p>(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.</p

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

<p>The cell stage was defined by soma size, process number and process diameter, number of branches and process length in relation to the soma diameter. The data for the table was the result of measuring between 150–200 cells per stage.</p