Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model

<p>Abstract</p> <p>Background</p> <p>Cardiac arrest, and the associated arrest of blood circulation, immediately leads to permanent brain damage because of the exhaustion of oxygen, glucose and energy resources in the brain. Most hippocampal CA1 neurons die during the first week post the insult. Molecular data concerning the recovery after resuscitation are sparse and limited to the early time period. Expression analysis of marker genes via quantitative real-time RT-PCR enables to follow up the remodeling process. However, proper validation of the applied normalization strategy is a crucial prerequisite for reliable conclusions.</p> <p>Therefore, the present study aimed to determine the expression stability of ten commonly used reference genes (<it>Actb</it>, actin, beta; <it>B2m</it>, beta-2 microglobulin;<it>CypA</it>, cyclophilin A; <it>Gapdh</it>, glyceraldehyde-3-phosphate dehydrogenase; <it>Hprt</it>, hypoxanthine guanine phosphoribosyl transferase; <it>Pgk1</it>, phosphoglycerate kinase 1; <it>Rpl13a</it>, ribosomal protein L13A; <it>Sdha</it>, succinat dehydrogenase complex, subunit a, flavoprotein (Fp); <it>Tbp</it>, TATA box binding protein; <it>Ywhaz</it>, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) in the rat hippocampus four, seven and twenty-one days after cardiac arrest. Moreover, experimental groups treated with the anti-inflammatory and anti-apoptotic drug minocycline have been included in the study as well.</p> <p>Results</p> <p>The microglial marker <it>Mac-1</it>, used as a target gene to validate the experimental model, was found to be upregulated about 10- to 20-fold after cardiac arrest.</p> <p>Expression stability of candidate reference genes was analyzed using geNorm and NormFinder software tools. Several of these genes behave rather stable. <it>CypA </it>and <it>Pgk1 </it>were identified by geNorm as the two most stable genes 4 and 21 days after asphyxial cardiac arrest, <it>CypA </it>and <it>Gapdh </it>at 7 days post treatment. <it>B2m </it>turned out to be the most variable candidate reference gene, being about 2-fold upregulated in the cardiac arrest treatment groups.</p> <p>Conclusion</p> <p>We have validated endogenous control genes for qRT-PCR analysis of gene expression in rat hippocampus after resuscitation from cardiac arrest. For normalization purposes in gene profiling studies a combination of <it>CypA </it>and <it>Pgk1 </it>should be considered 4 and 21 days post injury, whereas <it>CypA </it>and <it>Gapdh </it>is the best combination at 7 days. <it>CypA </it>is most favorable if restriction to a single reference gene for all time points is required.</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

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

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

Cytotoxicity of minocycline.

<p>MTT assay of glial cell cultures incubated with increasing minocycline concentrations (25, 50, 75, 100 and 125 µM) and incubation times (24, 48, 72 h and 7 days). Cytotoxicity of minocycline increased with incubation time and concentration. Statistical analysis was done with a 2-way ANOVA. Significant differences to the control are marked with * and demonstrate p<0.05. The assay was done in triples (n = 3 preparations) with a mean of 6 replicates each.</p

Motor neuron localization in organotypic spinal cord cultures.

<p><b>(A)</b> Displays the anatomy of spinal cord slices at lumbar level L5 modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073422#B46" target="_blank">46</a>]. Areas containing motor neurons are highlighted in grey and with a box. On the left, the spinal cord is shown as illustration; on the right side a corresponding anti-pan-NF stained spinal cord slice after 7 days of cultivation is shown. (<b>B</b> and <b>C</b>) show anti-GFAP stained astroglia in organotypic spinal cord cultures after 7 DIVs (C) magnifies stained astroglia marked with a box in (B). (<b>D</b> and <b>E</b>) illustrate anti-IBA-1 stained microglia in these cultures. (<b>E</b>) magnifies stained microglia marked with a box in (D).</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 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

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