Etablierung eines in vitro-Modells zur Untersuchung der Regeneration spinaler Motorneurone und ihrer Axone nach Axotomie und Neurodegeneration

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2015von Josephine Pinkernell

Magnetic nanoparticles in primary neural cell cultures are mainly taken up by microglia

<p>Abstract</p> <p>Background</p> <p>Magnetic nanoparticles (MNPs) offer a large range of applications in life sciences. Applications in neurosciences are one focus of interest. Unfortunately, not all groups have access to nanoparticles or the possibility to develop and produce them for their applications. Hence, they have to focus on commercially available particles. Little is known about the uptake of nanoparticles in primary cells. Previously studies mostly reported cellular uptake in cell lines. Here we present a systematic study on the uptake of magnetic nanoparticles (MNPs) by primary cells of the nervous system.</p> <p>Results</p> <p>We assessed the internalization in different cell types with confocal and electron microscopy. The analysis confirmed the uptake of MNPs in the cells, probably with endocytotic mechanisms. Furthermore, we compared the uptake in PC12 cells, a rat pheochromocytoma cell line, which is often used as a neuronal cell model, with primary neuronal cells. It was found that the percentage of PC12 cells loaded with MNPs was significantly higher than for neurons. Uptake studies in primary mixed neuronal/glial cultures revealed predominant uptake of MNPs by microglia and an increase in their number. The number of astroglia and oligodendroglia which incorporated MNPs was lower and stable. Primary mixed Schwann cell/fibroblast cultures showed similar MNP uptake of both cell types, but the Schwann cell number decreased after MNP incubation. Organotypic co-cultures of spinal cord slices and peripheral nerve grafts resembled the results of the dispersed primary cell cultures.</p> <p>Conclusions</p> <p>The commercial MNPs used activated microglial phagocytosis in both disperse and organotypic culture systems. It can be assumed that <it>in vivo </it>application would induce immune system reactivity, too. Because of this, their usefulness for <it>in vivo </it>neuroscientific implementations can be questioned. Future studies will need to overcome this issue with the use of cell-specific targeting strategies. Additionally, we found that PC12 cells took up significantly more MNPs than primary neurons. This difference indicates that PC12 cells are not a suitable model for natural neuronal uptake of nanoparticles and qualify previous results in PC12 cells.</p

Treatment of organotypic spinal cord cultures with 100 µM minocycline.

<p>Comparison of organotypic spinal cord cultures incubated with minocycline starting at DIV 1 or DIV 4 and analyzed at DIV 7. (A) Number of surviving motor neurons (anti-pan-NF stained). Minocycline incubation decreased this number significantly compared to controls. (B) Number of anti-NeuN stained neurons. Minocycline incubation reduced this number significantly compared to controls. (C) Percentage of anti-pan-NF stained area (neurofilaments). Minocycline incubation from DIV 1 onwards increased the percentage of neurofilament staining. Minocycline incubation from DIV 4 onwards, however, did not alter it. (D) Percentage of DAPI stained area. Minocycline incubation significantly decreased the DAPI cell nuclei staining in cultures incubated from DIV 1 and DIV 4 onwards. (E) Percentage of anti-IBA-1 stained area (microglia). Minocycline incubation reduced microglia pattern significantly compared to the control. (F) Anti-IBA-1 fluorescence staining intensity. Minocycline incubation reduced the fluorescence intensity of microglia in accordance with the reduced microglia spreading. (G) Percentage of anti-GFAP stained area (astroglia). Minocycline incubation did not influence the percentage of astroglia staining significantly if cultures were incubated from DIV 1 onwards. Incubation starting at DIV 4, however, reduced the percentage of astroglia staining significantly compared to controls. (H) Anti-GFAP fluorescence staining intensity. Minocycline incubation influenced fluorescence intensity of astroglia in accordance with the changes of area. If treatment started at DIV 1, fluorescence intensity was enhanced, whereas under treatment started at DIV4 a reduction was found. For statistical analysis a paired Student’s t-test was used to compare control and +Mino for each incubation period. Numbers of samples are: number of motor neurons and percentage of anti-pan-NF area: DIV 1 n=26, DIV 4 n=19; number of anti-NeuN stained neurons n=6 (DIV 1, DIV 4); percentage stained area of anti-IBA-1, anti-GFAP and DAPI: DIV 1 n=7, DIV 4n=6 (1 animal = mean of up to 3 replicates). All values are means ± standard deviation (SD). Significant differences are marked with * and demonstrate p<0.05.</p

Treatment of organotypic spinal cord co-cultures with 100 µM minocycline.

<p>Comparison of control and +Mino organotypic spinal cord co-cultures cultured for 7 days. +Mino co-cultures were incubated starting at DIV 1 or DIV 4. (A) number of motor neurons. Minocycline reduced the number of motor neurons compared to the control. (B) anti-pan-NF percentage stained area. Minocycline incubation from DIV 1 onwards resulted in an increased stained neurofilament respectively neurites area compared to the control. Incubation from DIV 4 onwards did not affect the percentage stained area. (C) anti-GFAP percentage stained area. (D) anti-GFAP fluorescence intensity. Neither astroglial percentage stained area nor fluorescence intensity was altered by minocycline. To compare controls and +Mino a paired Student’s t-test was used. Number of samples was n=6 except DIV 1 DAPI stained area with n=4 (1 animal = mean of up to 3 replicates). All values are means ± SD. Significant differences are marked with * and demonstrate p<0.05. (E–G) Fluorescence images of organotypic spinal cord co-cultures. A control culture is illustrated in (E) showing anti-pan-NF (green) stained motor neurons which grew neurites into the reconstructed ventral root. Astroglia (anti-GFAP, red) formed a glia cover on the surface of the slice. (F) demonstrates a co-culture incubated with minocycline from DIV 1 onwards lacking motor neurons and fiber outgrowth to the peripheral nerve graft that was opposed to the ventral side of the spinal cord slice (marked with grey <b>lines</b>). Astroglia staining shows peripheral distribution. (G) shows a co-culture treated from DIV 4 onwards. This culture shows several motor neurons, reconstruction of the ventral root and a glia cover. Bars = 500 µm.</p

Fluorescence images of spinal cord cultures treated with 100 µM minocycline.

<p>(A, D, G) controls; (B, E, H) incubation starting at DIV 1; (C, F, I) incubation from DIV 4 onwards. Slices were co-stained for anti-pan-NF (green) and anti-IBA-1 (red) in (A, B, C), for anti-pan-NF (green) and anti-GFAP (red) in (D, E, F) or for anti-NeuN (green) in (G, H, I). Motor neuronal areas are magnified in (A–F) and marked with a box (A–I). (A) Control cultures show various anti-pan-NF stained motor neurons, neurons, modest microglia activation, (D) glia cover formation and (G) anti-NeuN stained neuron populations. Cultures treated from DIV 1 onwards displayed (B) a lack of motor neurons and microglial staining, (E) a peripheral astroglial distribution and (H) a lack of anti-NeuN stained neurons. Cultures treated from DIV 4 onwards showed (C) less motor neurons and low microglia activation, (F) the formation of a glia cover on the slice surface and (I) some anti-NeuN stained neurons. Images (J–O) illustrate the development of the glia cover during cultivation. Slices are shown double stained with anti-pan-NF and anti-GFAP on the left and anti-GFAP staining only on the right. (J) displays a slice fixed after preparation (no cultivation). A peripheral anti-GFAP staining is visible. Slices were fixed and stained after 1 DIV (K), 2 DIV (L), 3 DIV (M), 5 DIV (N) or 7 DIV (O) of cultivation. The glial distribution changes over time from a peripheral distribution to a glia cover. Bars in (A–F, J–P) = 500 µm and in magnified area = 200 µm. Bars in (G–I) = 200 µm.</p

Western Blot analysis of primary glial cell cultures.

<p>Primary glial cell cultures were incubated for 72 h with 100 µM minocycline (+Mino). Control cells received medium without any supplement. Values in (A) are given as mean ratio of β-actin/GAPDH density ± SD and values in (C) are given as mean ratio of Cx43/GAPDH density ± SD. (<b>B</b>) shows representative images of β-actin bands of control and +Mino cells with corresponding GAPDH bands. (<b>D</b>) displays representative images of Cx43 bands of control and +Mino cells with corresponding GAPDH bands. Minocycline did not change the expression of β-actin but enhanced the expression of Cx43 compared to control cells. Statistical analysis was done with a paired Student’s t-test. A p-value ≤ 0.05 was considered to be statistically significant and is marked with *. The number of samples was n = 7.</p

Fluorescence images of spinal cord cultures treated with 10 µM minocycline.

<p>Fluorescent images of spinal cord cultures of control (A, D) and of slices incubated with 10 µM minocycline from DIV 1 (B, E) and from DIV 4 (C, F) onwards. Slices were co-stained for anti-pan-NF (green) and anti-IBA-1 (red) in (A, B, C) or for anti-pan-NF (green) and anti-GFAP (red) in (D, E, F) at DIV 7. Motor neuronal areas are magnified and marked with a box. Control cultures show various anti-pan-NF stained motor neurons, neurons, modest microglia activation (A) and formation of a glia cover (B). All cultures treated with minocycline show control-like staining patterns. No clear differences in the staining pattern are visible. Bars = 500 µm, bars in magnified area = 200 µm.</p

Scratching Assay of primary glial cell cultures.

<p>Cells were incubated with 100 µM minocycline for 72 h after wound scratching. All values in (A) are given as mean distance % of the starting distance of the wound ± SD. Closure of the wound was delayed in minocycline incubated cells compared to the control after 24 and 72 h. Images in (B) show phase contrast images of cultures with control cells and minocycline treated cells after 0, 24, 48 and 72 h incubation time. Wound distances are marked with black <b>lines</b>. Minocycline delayed wound closure compared to the control. Bars illustrate 250 µm. Statistical analysis was done with a paired Student’s t-test. Significant differences are marked with * and demonstrate p<0.05. The number of samples was n = 17 for 24 and 72 h and n=14 for 48 h.</p

Treatment of organotypic spinal cord cultures with 10 µM minocycline.

<p>Comparison of cultures incubated from DIV 1 or DIV 4 onwards and analyzed at DIV 7. (<b>A</b>) Number of surviving anti-pan-NF stained motor neurons. Minocycline incubation starting at DIV 1, but not at DIV 4, decreased the number of surviving motor neurons significantly. (<b>B</b>) <b>Percentage of anti-pan-NF stained area (neurofilaments). Minocycline incubation did not alter it</b>. (<b>C</b>) Percentage of anti-IBA-1 stained area (microglia). Minocycline incubation did not show a significant influence on microglia pattern. (<b>D</b>)Anti-IBA-1 fluorescence staining intensity. Early (long-term) minocycline incubation reduced the fluorescence intensity (activity) of microglia significantly. (<b>E</b>) Percentage of anti-GFAP stained area (astroglia). Minocycline incubation did not show an influence on astroglia pattern. (<b>F</b>) Anti-GFAP fluorescence staining intensity. Minocycline incubation did not show an influence on the fluorescence intensity (activity) of astroglia. (<b>G</b>) Percentage of DAPI stained area. Minocycline incubation increased the percentage area of DAPI cell nuclei staining in cultures incubated with minocycline from DIV 4, but not DIV 1 onwards. For statistical analysis a paired Student’s t-test was used to compare control and +Mino for each incubation period. Number of samples n=6 for each incubation time with minocycline (1 animal = mean of up to 3 replicates). All values are means ± SD. Significant differences are marked with * and demonstrate p<0.05.</p