Introduction

Pre-exposure of macrophages to LPS causes either tolerance, meaning that the responses to a second LPS-challenge are strongly attenuated, or priming, an elevated response to a second hit of LPS [1]. Cells or animals become LPS-tolerant when a first challenge with LPS causes a robust inflammatory response. Priming or sensitization is induced by ultra-low LPS-doses, that per se will not evoke substantial formation and release of pro-inflammatory cytokines. A state of LPS-tolerance can also be evoked in structures of the peripheral or central nervous system [2]. Whether or not LPS-sensitization will occur in a given neuroglial structure, especially in dorsal root ganglia (DRG), has not yet been investigated. The central goals of this study can, therefore, be summarized as follows: we first tried to determine ultra-low LPS-doses for a long-term stimulation of DRG cultures for 18 h, which per se did not cause elevations of TNF-α in the supernatants but an enhanced production of this cytokine by a second hit with a high LPS-dose (“sensitization”). We further tested the effects of the presence of ultra-low, moderate and high LPS-doses on the capsaicin-evoked responses of neurons from rat DRG.

Materials and methods

See supplementary material.

Results

Cultivation of DRG primary cultures in presence of various doses of LPS was accompanied by a dose dependent rise of TNF-α in the supernatants (Fig. 1A). The lowest LPS-doses used in this experiment (0.01 and 0.001 µg/ml) did not evoke a significant increase of TNF-α in the supernatants. To test whether primary DRG cultures were sensitized to a subsequent hit with a high LPS dose, cultures were stimulated with 10 µg/ml LPS for 2 h after pre-exposure with 0.1, 0.01 or 0.001 µg/ml LPS for 18 h (Fig. 1B). The slight LPS-induced (10 µg/ml) increase of TNF-α in supernatants of cells pre-treated with 0.001 µg/ml LPS was not significant.

Fig. 1
figure 1

A, B TNF-α release after incubation with different doses of LPS and PBS: DRG primary cultures were incubated with different doses of LPS (0.001, 0.01, 0.1, 1 µg/ml) for 18 h or PBS as control (A) for subsequent measurement of TNF-α in the supernatants. In a second series of experiments (B), DRG primary cultures were pre incubated with different doses (0.001, 0.01, 0.1 µg/ml) for 18 h as well as PBS as control followed by a second stimulation with a high LPS dose (10 µg/ml) for 2 h. Pre-incubation with 0.001 μg/ml followed by a stimulation with a high LPS dose showed a tendency of a higher TNF-α release into supernatants compared to control. Each column represents the mean ± SEM of n samples and four different experiments. One-way ANOVA followed by a Newman–Keuls multiple comparison test was performed for statistical analysis. The graphical depiction of the p values were illustrated as follows: ***p < 0.001; **p < 0.01; *p < 0.05; D DRG primary cells were stimulated with different doses (0.001, 0.01, 0.1, 1 μg/ml) of LPS for 18 h and PBS as control. KCl served as vitality control for DRG neurons. ∆ratio [340/380 nm] represents the mean ± SEM increase of intracellular calcium [Ca2+]i of n cells of six different preparations. Percentages represent the numbers of responsive cells to a distinct stimulus, e.g., capsaicin compared to all vital neurons (KCl). Statistical analysis of KCl or capsaicin responses was performed using a one-way ANOVA followed by a Newman–Keuls multiple comparison test. p values were represented as follows: ***p < 0.001; **p < 0.01; *p < 0.05; C, E Prior to the experiments DRG primary cultures were incubated with LPS (1 μg/ml for 18 h) or PBS (18 h). After incubation different stimuli (capsaicin [10−7 M], KCl and capsaicin [10–6 M]) were applied to investigate neuronal responses. C Examples of tracings recorded from three DRG neurons: one neuron responding to KCl [50 mM] only (green), one responding to KCl and capsaicin [10−6 M] (red) and one responding to all three stimuli KCl, capsaicin [10−6 M and 10−7 M] (blue). E Depicts ∆ratio [340/380 nm] fluorescence values as a measurement of [Ca2+]. Columns represent the mean ± SEM increase of intracellular calcium [Ca2+]i of n cells from 4 different preparations. Statistical analysis between the PBS and LPS group was performed using an unpaired t test. ***p < 0.001 (color figure online)

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DRG primary cultures were incubated in presence of PBS or LPS at various doses (0.001, 0.01, 0.1 or 1 µg/ml) for 18 h. Thereafter, the strength of stimulus-induced Ca2+-signals of DRG-neurons was evaluated [3]. Capsaicin, at a dose of 10–6 M, evoked pronounced Ca2+-responses in 60–70% of neurons from all groups (Fig. 1D).

The responses of neurons to the depolarizing KCl-solution (vitality-test) was similar in all groups investigated. A significant enhancement of the strength of capsaicin-induced Ca2+-signals was exclusively determined in the group, which was pre-treated with the highest LPS-dose (1 µg/ml, Fig. 1D), the same dose, which evoked a profound increase of TNF-α in the supernatants (Fig. 1A). A sensitization of DRG neurons to a nociceptive stimulus (capsaicin) [4] thus was not achieved by pre-treatment with very low LPS-dose, which per se did not evoke an increase of TNF-α production.

We finally tested whether DRG neurons might also show enhanced responses to the threshold-dose of capsaicin. We determined that not a single DRG neuron showed Ca2+-responses to capsaicin at doses 10–9 and 10–8 M. Stimulation with 10–7 M capsaicin evoked Ca2+-signals just in single neurons. This dose was, therefore, defined as the threshold dose (Fig. 1C).

Just 4 out of 231 DRG neurons (about 2%) responded to 10–7 M capsaicin. In cultures pre-treated with 1 µg/ml LPS 8 out of 223 neurons (about 4%) were responsive to the capsaicin threshold dose (Fig. 1D). Vitality of neurons (responses to KCl) was identical in both groups. Again, the responses of DRG neurons to the effective capsaicin-dose of 10–6 M was significantly higher in neurons pre-exposed to 1 µg/ml LPS.

Discussion

Priming of macrophages with a sub-threshold dose of LPS resulted in enhanced release of TNF-α to a second stimulation with a higher LPS-dose [5]. This effect seems to be the basic mechanism for the phenomenon of LPS-sensitization. We aimed to define experimental conditions, which should mimic such an effect in mixed neuro-glial primary cultures from rat DRG. With regard to the formation and release of TNF-α, the outcome was not as clear as we expected. When pre-treated with an ultra-low LPS-dose, an enhanced TNF-α response to a subsequent stimulation with a high LPS-dose was hardly detectable. Still, there is evidence for a sensitization of capsaicin-responsive (nociceptive) sensory neurons from thoracic DRG with LPS resulting in hypersecretion in the upper airways [6]. Therefore, we tested the effects of cultivation of DRG primary cultures in presence of various doses of LPS on the strength of capsaicin-evoked Ca2+-signals. To evoke enhanced capsaicin responses, the presence of an amount of LPS in the culture medium is required, which induces a robust release of TNF-α into the supernatant. Sub-threshold doses of LPS failed to induce such an effect. This means that a given inflammatory insult has to reach a certain threshold to cause a sensitization of peripheral nociceptors finally resulting in the manifestation of inflammatory pain. Our observation that ultra-low doses of LPS failed to evoke sensitization of DRG nociceptive neurons (capsaicin-responses) and the mixed neuroglial culture (formation of TNF-α) can possibly be explained by the fact that just 2% of all cells of DRG cultures are macrophages [2]. Cells from the macrophage–monocyte-lineage seem to be critical for sensitization to LPS, also in structures of the nervous system [7, 8]. Future studies should, therefore, employ central nervous structures including a higher percentage of cells from the macrophage–monocyte-lineage to investigate the phenomenon of LPS-sensitization in mixed neuroglial tissue.