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. 2010 Dec 27:6:98.
doi: 10.1186/1744-8069-6-98.

Quantitative automated microscopy (QuAM) elucidates growth factor specific signalling in pain sensitization

Christine Andres  1 , Sonja Meyer, Olayinka A Dina, Jon D Levine, Tim Hucho
Affiliations

Quantitative automated microscopy (QuAM) elucidates growth factor specific signalling in pain sensitization

Christine Andres et al. Mol Pain. .

Abstract

Background: Dorsal root ganglia (DRG)-neurons are commonly characterized immunocytochemically. Cells are mostly grouped by the experimenter's eye as "marker-positive" and "marker-negative" according to their immunofluorescence intensity. Classification criteria remain largely undefined. Overcoming this shortfall, we established a quantitative automated microscopy (QuAM) for a defined and multiparametric analysis of adherent heterogeneous primary neurons on a single cell base.The growth factors NGF, GDNF and EGF activate the MAP-kinase Erk1/2 via receptor tyrosine kinase signalling. NGF and GDNF are established factors in regeneration and sensitization of nociceptive neurons. If also the tissue regenerating growth factor, EGF, influences nociceptors is so far unknown. We asked, if EGF can act on nociceptors, and if QuAM can elucidate differences between NGF, GDNF and EGF induced Erk1/2 activation kinetics. Finally, we evaluated, if the investigation of one signalling component allows prediction of the behavioral response to a reagent not tested on nociceptors such as EGF.

Results: We established a software-based neuron identification, described quantitatively DRG-neuron heterogeneity and correlated measured sample sizes and corresponding assay sensitivity. Analysing more than 70,000 individual neurons we defined neuronal subgroups based on differential Erk1/2 activation status in sensory neurons. Baseline activity levels varied strongly already in untreated neurons. NGF and GDNF subgroup responsiveness correlated with their subgroup specificity on IB4(+)- and IB4(-)-neurons, respectively. We confirmed expression of EGF-receptors in all sensory neurons. EGF treatment induced STAT3 translocation into the nucleus. Nevertheless, we could not detect any EGF induced Erk1/2 phosphorylation. Accordingly, intradermal injection of EGF resulted in a fundamentally different outcome than NGF/GDNF. EGF did not induce mechanical hyperalgesia, but blocked PGE2-induced sensitization.

Conclusions: QuAM is a suitable if not necessary tool to analyze activation of endogenous signalling in heterogeneous cultures. NGF, GDNF and EGF stimulation of DRG-neurons shows differential Erk1/2 activation responses and a corresponding differential behavioral phenotype. Thus, in addition to expression-markers also signalling-activity can be taken for functional subgroup differentiation and as predictor of behavioral outcome. The anti-nociceptive function of EGF is an intriguing result in the context of tissue damage but also for understanding pain resulting from EGF-receptor block during cancer therapy.

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Figures

Figure 1
Figure 1
Automatic DRG-neuron identification and quantification of phosphospecific fluorescence intensities. A) Example image of a microscope view field of the DRG neuron culture immunofluorescently labelled for the neuronal marker PGP9.5. Neurons show high degree of heterogeneity in respect to size, fluorescence intensity and extend of cell-cell contacts. B) Examples of objects selected as neurons (upper two rows) and rejected objects (lower two rows). Rejected objects include non-cellular debris, glia cells and clustered neurons. C) "Virtual wells" were created by randomly selecting 250, 1000, 5000, 10,000 or 20,000 cells per virtual well out of 49,503 measured cells. The normalized mean well intensity of 10,000 such virtual wells with the respective standard deviation is depicted indicating the sensitivity limit of studies based on the various average cell numbers per well (250 cells per well resulted in a well Mean Intensity of 1.0005 ± 0.1133, 1000 cells in 1.0003 ± 0.0564, 5000 cells in 0.9999 ± 0.0240, 10,000 cells in 0.9997 ± 0.0159, and 20,000 cells result 0.9998 ± 0.0098). D) IB4 lectin derived fluorescence intensities showed a continuous distribution of intensities. Coincubation with uncoupled lectin to block specific binding resulted in an intensity histogram (n = 5002 cells, black line) matching the first peak of the unblocked IB4-intensity histogram (n = 23266 cells, red line) identifying these cells as unspecificly binding and thus as IB4(-)-cells. E) Diameter histogram of 49,503 neurons (yellow bars). 40,706 neurons were labeled for IB4. IB4(+)-neurons (red bars) show a higher average diameter than IB4(-)-neurons (blue bars). F) Comparison of the normalized Mean Intensity of phosphorylated and total Erk1/2 in IB4(+)- and IB4(-)-neurons (n = 1421 IB4(+)-neurons and n = 963 IB4(-)-neurons). IB4(+)-neurons show a higher amount of pErk1/2 but also a higher expression rate of Erk1/2 in comparison to IB4(-)-neurons (p < 0.001).
Figure 2
Figure 2
NGF and GDNF stimulation leads to Erk1/2 phosphorylation in subpopulations. A) Stimulation with NGF (1 nM, 30 min) and GDNF (1 nM, 30 min) led to significant increase of pErk1/2 intensity levels (n = 5203 cells for no stimulus, n = 3778 cells for NGF treatment, n = 1275 cells for GDNF treatment, error bars are SEM, p < 0.001 for NGF/GDNF vs control). B) 1D scatter plot of the single cell data plotted in A) as bar graphs. Thereby, the huge heterogeneity with up to 100-fold intensity differences is clearly visible. C) Intensity histogram of unstimulated (black line), NGF (red line), GDNF (blue line) and PMA (orange line) stimulated DRG-cultures. Stimulation led to increased numbers of cells with higher fluorescent intensities. PMA activates Erk1/2 in virtually all neurons as nearly no cells remain with intensities of the control condition. D), E) Cell size profile of NGF/GDNF responding and non-responding cells. Responding neurons tend to be smaller in size. F), G) IB4 fluorescence intensity profile of NGF/GDNF responding and non-responding cells. NGF responding neurons tend to low intensity IB4 labeling while GDNF responding cells are mostly strongly IB4 labeled. H) Kinetic of NGF/GDNF induced Erk1/2 phosphorylation (n = 1000-4000 cells per time point, error bars SEM).
Figure 3
Figure 3
EGFR is expressed in DRG-neurons. A) Analysis of EGFR mRNA expression in lysates of DRG and brain from male rats by RT-PCR. RT-PCR shows a DNA-fragment with increasing intensity in dependence of the number (30, 35 and 40) of amplification cycles for DRG (lane 1-3) and brain (lane 6-8). Lane 4 and 9 reaction without reverse transcriptase (-RT) and lane 5 and 10 water control. B) Confocal images of DRG-sections. Left panel EGFR-staining, middle panel EGFR + blocking peptide, right panel secondary antibody control leaving out the primary antibody. C-terminal EGFR antibody blocking peptide was used in a 100× higher concentration than the antibody. C) Confocal images of DRG-cultures. Left panel EGFR-staining, middle panel EGFR + peptide, right panel secondary control. C-terminal EGFR antibody blocking peptide was used in a 100× higher concentration than the antibody. Intensity profiles along the indicated line crossing the cells indicate the plasma membrane staining of the EGFR-antibody. EGFR-C-terminal blocking peptide abolished the plasma membrane EGFR-signal D) QuAM quantified intensity histogram of EGFR-stained cultures (red), EGFR + blocking peptide (blue) and secondary control (black) indicated that EGFR is expressed in all neurons (n = 1874 cell for secondary control, n = 1720 cells EGFR, n = 1705 cells for EGFR + blocking peptide; p < 0.001).
Figure 4
Figure 4
EGF induces STAT3 translocation but not Erk1/2 phosphorylation. A) Kinetic of phosphorylation of Erk1/2 in response to 1 nM EGF treatment of DRG-neurons (n = 1000-2000 cells for each time point, error bars SEM). B) Dose response curve after 5 min or 10 min stimulation (n = 1000-3000 cells for each concentration, error bars SEM). C) 1 nM EGF induced phosphorylation and translocation of STAT3 into the nucleus of DRG-neurons. D) Quantification of neurons showing phosphorylated STAT3 in the nucleus in response to 1 nM EGF (p < 0.01; n = 300 cells per condition were counted in 3 independent experiments). E) Mean intensity of pSTAT3 increased in nuclei of neurons in response to 1 nM EGF treatment (p < 0.01; Mean intensity of nuclei (61 (control) and 83 (EGF treated)) were quantified in 3 independent experiments).
Figure 5
Figure 5
EGF blocks PGE2 induced mechanical hyperalgesia. Intradermal injection of 1 μg EGF and 1 μg Hb-EGF led not to a significant decrease in the nociceptive threshold in contrast to intradermal injection of 100 ng PGE2 or 100 ng Epinephrine. EGF and Hb-EGF abolished subsequent PGE2 but not epinephrine induced sensitization (p < 0.001). Reduction of paw-withdrawal threshold in EGF/PGE2 data series by 3.6 ± 1.9% after EGF treatment, 33.0 ± 3.8% after PGE2 treatment, 2.6 ± 1.3% after EGF+PGE2. (n = 6). Reduction of paw-withdrawal threshold in EGF/epinephrine data series by 8.5 ± 4.1% after EGF treatment, 36.2 ± 1.8% after epinephrine and 35.9 ± 1.5% after EGF+epinephrine (for all treatments n = 6). Reduction of paw-withdrawal threshold in Hb-EGF data series (3.0 ± 1.8% after Hb-EGF (n = 12), 36.0 ± 0.8% after PGE2 treatment, 3.6 ± 2.9% after Hb-EGF+PGE2, 38.6 ± 2.9% after epinephrine and 35.6 ± 1.7% after Hb-EGF+epinephrine (for all treatments n = 6)).
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