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. 2017 Feb 27;12:1577–1591. doi: 10.2147/IJN.S127206

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© 2017 Pohland et al. This work is published and licensed by Dove Medical Press Limited

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DMDP treatment decreased the amount of microglia and altered OHSC viability.

Notes: VSOP treatment did not change chemokine and cytokine homeostasis but contributed with and without microglia depletion to increasing neuronal cell death. (A–D) Confocal images of the microglia depletion experiments. Scale bar =300 μm. (A) Iba1-microglia staining (red) of the control slice without depletion; CA, DG, SUB. (B) Microglia labeling of the control slice with depletion shows decreased amount of Iba1-positive cells compared to (A). (C) GFAP-astrocyte (green) staining of the control slice with microglia depletion shows maintained cellular astrocytic organization. (D) Merge of (B) and (C). (E–R) PI fluorescence images of representative hippocampal slices shown in inverted colors. The first and third columns of the pictures were taken after 7 days of equilibration time post preparation and 2 more days with or without microglia depletion until 9 DIV. The second and fourth columns of the images were photographed after 2 more days of incubation using identical slices and treatment with or without 3.0 mM VSOP-R1 and VSOP-R2 or 10 mM of Glut until 11 DIV. Scale bar =500 μm. (E) Same hippocampal slice before and (F) after 3.0 mM VSOP-R1 treatment. (G) Identical hippocampal slice with depleted microglia before and (H) after 3.0 mM VSOP-R1 treatment. (I) Same hippocampal slice before and (J) after 3.0 mM VSOP-R2 treatment. (K) Identical hippocampal slice with depleted microglia before and (L) after 3.0 mM VSOP-R2 treatment. (M) Same negative control without VSOP treatment at 9 DIV and 11 DIV (N). (O) Identical control with microglia depletion without VSOP treatment at 9 DIV and 11 DIV (P). (Q) Same positive control with 10 mM Glut treatment at 9 DIV and 11 DIV (R). (S–U) Integrated PI fluorescence intensities of hippocampal slices with or without microglia depletion before (9 DIV) and after (11 DIV) 3.0 mM VSOP-R1 or VSOP-R2 treatment and Glut incubation. CA1, CA3, and DG regions were analyzed. (S) Microglia depletion with and without VSOP-R1 incubation affects hippocampal CA1 viability but not VSOP-R2 alone (error bars represent SD, number of slices: n=12 for untreated control; n=17 for DMDP; n=18 for 3.0 mM VSOP-R1; n=18 for 3.0 mM VSOP-R1 + DMDP; n=17 for 3.0 mM VSOP-R2; n=18 for 3.0 mM VSOP-R2 + DMDP; n=6 for Glut). (T) Microglia depletion with and without VSOP-R1/R2 incubation alters hippocampal CA3 viability (error bars represent SD, number of slices: n=12 for untreated control; n=17 for DMDP; n=18 for 3.0 mM VSOP-R1; n=18 for 3.0 mM VSOP-R1 + DMDP; n=17 for 3.0 mM VSOP-R2; n=18 for 3.0 mM VSOP-R2 + DMDP; n=6 for Glut). (U) Microglia depletion with and without VSOP-R1 incubation affects hippocampal DG viability but not VSOP-R2 alone (error bars represent SD, number of slices: n=12 for untreated control; n=17 for DMDP; n=18 for 3.0 mM VSOP-R1; n=18 for 3.0 mM VSOP-R1 + DMDP; n=17 for 3.0 mM VSOP-R2; n=18 for 3.0 mM VSOP-R2 + DMDP; n=6 for Glut). (V) Summarized statistical analysis of (S–U); NS = not significant, **P<0.01, and ***P<0.001 were considered significant. (W–Z) Hippocampal slice culture cytokine secretion analysis measured after VSOP treatment with or without microglia depletion in the supernatant at 11 DIV. (W) Microglia depletion with and without VSOP incubation did not affect CXCL1/KC secretion (error bars represent SD, number of supernatant double determinations for all groups: n=4; ***P<0.001 was considered significant). (X) Microglia depletion with and without VSOP treatment did not alter IL-6 secretion (error bars represent SD, number of supernatant double determinations for all groups: ***P<0.001 was considered significant). (Y) Microglia depletion with and without VSOP incubation did not affect MCP-1 secretion (error bars represent SD, number of supernatant double determinations for all groups: n=4; ***P<0.001 was considered significant). (Z) Microglia depletion with and without VSOP treatment did not alter TNF-alpha secretion (error bars represent SD, number of supernatant double determinations for all groups: n=4; ***P<0.001 was considered significant).

Abbreviations: CA, cornu ammonis; CXCL1, C-X-C motif chemokine; DG, dentate gyrus; DIV, days in vitro; DMDP, dichloromethylenediphosphonic acid disodium salt; GFAP, glial fibrillary acidic protein; Glut, glutamate; GM-CSF, granulocyte-macrophage colony-stimulating factor; Iba1, ionized calcium binding adapter molecule 1; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; Neg, negative; OHSC, organotypic hippocampal slice culture; PI, propidium iodide; Pos, positive; SD, standard deviation; SUB, subiculum; TNF, tumor necrosis factor; VSOP, very small superparamagnetic iron oxide particles.

DMDP treatment decreased the amount of microglia and altered OHSC viability.

Notes: VSOP treatment did not change chemokine and cytokine homeostasis but contributed with and without microglia depletion to increasing neuronal cell death. (A–D) Confocal images of the microglia depletion experiments. Scale bar =300 μm. (A) Iba1-microglia staining (red) of the control slice without depletion; CA, DG, SUB. (B) Microglia labeling of the control slice with depletion shows decreased amount of Iba1-positive cells compared to (A). (C) GFAP-astrocyte (green) staining of the control slice with microglia depletion shows maintained cellular astrocytic organization. (D) Merge of (B) and (C). (E–R) PI fluorescence images of representative hippocampal slices shown in inverted colors. The first and third columns of the pictures were taken after 7 days of equilibration time post preparation and 2 more days with or without microglia depletion until 9 DIV. The second and fourth columns of the images were photographed after 2 more days of incubation using identical slices and treatment with or without 3.0 mM VSOP-R1 and VSOP-R2 or 10 mM of Glut until 11 DIV. Scale bar =500 μm. (E) Same hippocampal slice before and (F) after 3.0 mM VSOP-R1 treatment. (G) Identical hippocampal slice with depleted microglia before and (H) after 3.0 mM VSOP-R1 treatment. (I) Same hippocampal slice before and (J) after 3.0 mM VSOP-R2 treatment. (K) Identical hippocampal slice with depleted microglia before and (L) after 3.0 mM VSOP-R2 treatment. (M) Same negative control without VSOP treatment at 9 DIV and 11 DIV (N). (O) Identical control with microglia depletion without VSOP treatment at 9 DIV and 11 DIV (P). (Q) Same positive control with 10 mM Glut treatment at 9 DIV and 11 DIV (R). (S–U) Integrated PI fluorescence intensities of hippocampal slices with or without microglia depletion before (9 DIV) and after (11 DIV) 3.0 mM VSOP-R1 or VSOP-R2 treatment and Glut incubation. CA1, CA3, and DG regions were analyzed. (S) Microglia depletion with and without VSOP-R1 incubation affects hippocampal CA1 viability but not VSOP-R2 alone (error bars represent SD, number of slices: n=12 for untreated control; n=17 for DMDP; n=18 for 3.0 mM VSOP-R1; n=18 for 3.0 mM VSOP-R1 + DMDP; n=17 for 3.0 mM VSOP-R2; n=18 for 3.0 mM VSOP-R2 + DMDP; n=6 for Glut). (T) Microglia depletion with and without VSOP-R1/R2 incubation alters hippocampal CA3 viability (error bars represent SD, number of slices: n=12 for untreated control; n=17 for DMDP; n=18 for 3.0 mM VSOP-R1; n=18 for 3.0 mM VSOP-R1 + DMDP; n=17 for 3.0 mM VSOP-R2; n=18 for 3.0 mM VSOP-R2 + DMDP; n=6 for Glut). (U) Microglia depletion with and without VSOP-R1 incubation affects hippocampal DG viability but not VSOP-R2 alone (error bars represent SD, number of slices: n=12 for untreated control; n=17 for DMDP; n=18 for 3.0 mM VSOP-R1; n=18 for 3.0 mM VSOP-R1 + DMDP; n=17 for 3.0 mM VSOP-R2; n=18 for 3.0 mM VSOP-R2 + DMDP; n=6 for Glut). (V) Summarized statistical analysis of (S–U); NS = not significant, **P<0.01, and ***P<0.001 were considered significant. (W–Z) Hippocampal slice culture cytokine secretion analysis measured after VSOP treatment with or without microglia depletion in the supernatant at 11 DIV. (W) Microglia depletion with and without VSOP incubation did not affect CXCL1/KC secretion (error bars represent SD, number of supernatant double determinations for all groups: n=4; ***P<0.001 was considered significant). (X) Microglia depletion with and without VSOP treatment did not alter IL-6 secretion (error bars represent SD, number of supernatant double determinations for all groups: ***P<0.001 was considered significant). (Y) Microglia depletion with and without VSOP incubation did not affect MCP-1 secretion (error bars represent SD, number of supernatant double determinations for all groups: n=4; ***P<0.001 was considered significant). (Z) Microglia depletion with and without VSOP treatment did not alter TNF-alpha secretion (error bars represent SD, number of supernatant double determinations for all groups: n=4; ***P<0.001 was considered significant).

Abbreviations: CA, cornu ammonis; CXCL1, C-X-C motif chemokine; DG, dentate gyrus; DIV, days in vitro; DMDP, dichloromethylenediphosphonic acid disodium salt; GFAP, glial fibrillary acidic protein; Glut, glutamate; GM-CSF, granulocyte-macrophage colony-stimulating factor; Iba1, ionized calcium binding adapter molecule 1; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; Neg, negative; OHSC, organotypic hippocampal slice culture; PI, propidium iodide; Pos, positive; SD, standard deviation; SUB, subiculum; TNF, tumor necrosis factor; VSOP, very small superparamagnetic iron oxide particles.