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

Visual Response Properties of Y Cells in the Detached Feline Retina Visual Neurophysiology

PURPOSE. To evaluate early changes in the visual response properties of Y cells in the detached feline retina. METHODS. The retinas of young adult cats were detached by injection, with a glass micropipette, of a solution of 0.004% sodium hyaluronate in a balanced salt solution between the neural retina and the retinal pigment epithelium. At 1, 3, and 7 days after detachment, the eyes were removed. The eyecup was prepared as a flat mount in a recording chamber and superfused with medium. Extracellular single-unit responses from Y cells in the retinas were recorded. RESULTS. One, 3, and 7 days after retinal detachment surgery, Y cells showed clear signs of functional deterioration. At each time point, more ON center cells than OFF cells were encountered. Y cells in the detached retinas showed a statistically significant elevation in the average threshold irradiance after 1-, 3-, and 7-day detachment, respectively. The average contrast threshold recorded from cells in the normal retina was 3.6%, but it increased to 14.5%, 21.8%, and 47.5% after 1-, 3-, and 7-day detachment, respectively. Furthermore, at each time point, the capability of Y cells to process contrast information decreased significantly more because of detachment than because of luminance task performance. CONCLUSIONS. Retinal detachment induced rapid functional remodeling that resulted in degenerated Y-cell function, including an elevated luminance threshold and a deteriorated contrast threshold. Detachment had a greater impact on the latter. These physiological changes after retinal detachment could be used as objective indicators of early deterioration of visual function in future studies of retinal remodeling. (Invest Ophthalmol Vis Sci. 2010;51:1208 -1215 This is partly attributed to the intrinsic nature of photoreceptor cells, in that outer segments are able to regenerate after reattachment and that most of them achieve near-normal morphologies. Nevertheless, visual deficits are common after successful reattachment surgery. 7-9 Regardless of the initial insult, stressed photoreceptors can remodel their synaptic terminals and their relationship to second-order neurons. These changes are proposed to progress through three phases: photoreceptor stress, photoreceptor death, and complex neural remodeling. 10 Although the cells are in detachment, neural remodeling occurs early-within a few days-and, therefore, before massive photoreceptor cell death. Substantial evidence suggests that diagnosis and intervention at the early stages of retinal remodeling, especially during the stress phase, when there is significant photoreceptor terminal modifications, are critical for the successful rescue of injured cells and the restoration of visual function. Over the years, molecular and morphologic changes during this stage have been explicitly addressed in experimental retinal detachment, 3 whereas objective neurophysiological observations correlating these early structural changes have been reported only in data from electroretinography. 11 Electroretinographic (ERG) testing provides one objective measurement of the electrical activity of detached and reattached retinas. 12-16 However, current ERG technology is not sensitive enough to detect alterations at the single-cell level. RGCs play the crucial role in collecting appropriate input from second-order neurons and transmitting visual information to higher visual centers, but little is known about the functional consequence of these neurons after RD and subsequent reattachment. Y cells have been studied for more than 40 years, From th

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.

<p>The blue bar is the net direction of the processes.</p

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

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

<p>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).</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

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).

<p>(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).</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

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