Department for Molecular Human Genetics, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, Berlin, Germany.
Mol Pain. 2010 Dec 27;6:98. doi: 10.1186/1744-8069-6-98.
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.
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.
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.
背根神经节（DRG）神经元通常通过免疫细胞化学进行特征描述。根据免疫荧光强度，细胞主要由实验者的眼睛分为“标记阳性”和“标记阴性”。分类标准仍然很大程度上没有定义。为了克服这一不足，我们建立了一种定量自动显微镜（QuAM），用于对贴壁异质原代神经元进行定义和多参数分析，其基础是单细胞。生长因子 NGF、GDNF 和 EGF 通过受体酪氨酸激酶信号转导激活 MAP 激酶 Erk1/2。NGF 和 GDNF 是痛觉神经元再生和敏化的既定因素。组织再生生长因子 EGF 是否也影响伤害感受器目前尚不清楚。我们想知道 EGF 是否可以作用于伤害感受器，以及 QuAM 是否可以阐明 NGF、GDNF 和 EGF 诱导的 Erk1/2 激活动力学之间的差异。最后，我们评估了是否可以通过研究一个信号成分来预测对未在伤害感受器上测试的试剂（如 EGF）的行为反应。
我们建立了一种基于软件的神经元识别方法，定量描述了 DRG 神经元的异质性，并将测量的样本量和相应的检测灵敏度相关联。分析了超过 70,000 个单个神经元，我们根据感觉神经元中 Erk1/2 激活状态的差异定义了神经元亚群。未经处理的神经元中，基线活性水平差异很大。NGF 和 GDNF 亚群的反应性分别与其在 IB4(+)-和 IB4(-)-神经元上的亚群特异性相关。我们证实所有感觉神经元均表达 EGF 受体。EGF 处理诱导 STAT3 易位入核。然而，我们未能检测到任何 EGF 诱导的 Erk1/2 磷酸化。因此，皮内注射 EGF 的结果与 NGF/GDNF 根本不同。EGF 不会诱导机械性痛觉过敏，但会阻断 PGE2 诱导的敏化。
QuAM 是分析异质培养物中内源性信号转导激活的合适工具（如果不是必需的话）。DRG 神经元的 NGF、GDNF 和 EGF 刺激显示出不同的 Erk1/2 激活反应和相应的不同行为表型。因此，除了表达标志物外，信号活性也可用于功能亚群分化，并作为行为结果的预测指标。EGF 的抗伤害感受功能在组织损伤的背景下是一个有趣的结果，但对于理解癌症治疗期间 EGF 受体阻断引起的疼痛也很重要。
