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. 2015 Jan;16(1):60-6.
doi: 10.1016/j.jpain.2014.10.007. Epub 2014 Nov 4.

Plasma membrane mechanisms in a preclinical rat model of chronic pain

Luiz F Ferrari  1 , Jon D Levine  2
Affiliations

Plasma membrane mechanisms in a preclinical rat model of chronic pain

Luiz F Ferrari et al. J Pain. 2015 Jan.

Abstract

We have recently shown that the prolongation of prostaglandin E2 hyperalgesia in a preclinical model of chronic pain-hyperalgesic priming-is mediated by release of cyclic adenosine monophosphate from isolectin B4-positive nociceptors and its metabolism by ectonucleotidases to produce adenosine. The adenosine, in turn, acts in an autocrine mechanism at an A1 adenosine receptor whose downstream signaling mechanisms in the nociceptor are altered to produce nociceptor sensitization. We previously showed that antisense against an extracellular matrix molecule, versican, which defines the population of nociceptors involved in hyperalgesic priming, eliminated the prolongation of prostaglandin E2 hyperalgesia. To further evaluate the mechanisms at the interface between the extracellular matrix and the nociceptor's plasma membrane involved in hyperalgesia prolongation, we interrupted a plasma membrane molecule involved in versican signaling, integrin β1, with an antisense oligodeoxynucleotide. Integrin β1 antisense eliminated mechanical hyperalgesia induced by an adenosine A1 receptor agonist, cyclopentyladenosine, in the primed rat. We also disrupted a molecular complex of signaling molecules that contains integrin β1, lipid rafts, with methyl-β-cyclodextrin, which attenuated the prolongation without affecting the acute phase of prostaglandin E2 hyperalgesia, while having no effect on cyclopentyladenosine hyperalgesia. Our findings help to define the plasma membrane mechanisms involved in a preclinical model of chronic pain.

Perspective: The present study contributes to a further understanding of mechanisms involved in the organization of messengers at the plasma membrane that participate in the transition from acute to chronic pain.

Keywords: Extracellular matrix; hyperalgesia; hyperalgesic priming; integrin β1; nociceptor; rat; versican.

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Figures

Figure 1
Figure 1. Role of integrin β1 in expression of hyperalgesic priming
The schematic on the top of the figure represents the protocol used in the experiment, with the arrowheads indicating the time points when the rat hind paw mechanical threshold was evaluated. (A) The PKCε activator, ψεRACK (1 μg), was injected intradermally on the dorsum of the hind paw and, the mechanical nociceptive threshold, evaluated 30 min, 4 h and 10 days later. Significant mechanical hyperalgesia was observed at 30 min and 4 h, and by the 10th day after injection, the mechanical thresholds were not significantly different from pre-ψεRACK values; (B) Rats were then treated intrathecally, from days 11 to 13 post-ψεRACK, with ODN-AS (black bars) or -MM (white bars) for integrin β1 mRNA. On day 14, CPA (1 μg) was injected at the same site where ψεRACK had previously been administered and, the mechanical nociceptive threshold, evaluated 30 min and 4 h later. No hyperalgesia was observed in the group treated with ODN-AS, as opposed to the MM group (F1,10=70.74; ***p<0.0001 when comparing both groups; two-way repeated measures ANOVA followed by Bonferroni post-test, n = 6 paws per group), suggesting that the expression of priming (i.e., induction of mechanical hyperalgesia by CPA) may be dependent on the presence of integrin β1 at the peripheral terminal of the nociceptor.
Figure 2
Figure 2. Role of lipid rafts in the expression of hyperalgesic priming
The time lines on the top of the graphs show the protocols used in the respective experiments. Panel A: Rats previously (2 weeks) primed with intradermal injection of the PKCε activator ψεRACK (1 μg; not shown) on the dorsum of the right hind paw received an injection, at the same site, of vehicle (white bars) or the lipid raft disruptor methyl-β-cyclodextrin (MβC, 1 μg, black bars). 15 min later, PGE2 was injected and, the mechanical hyperalgesia, evaluated 30 min and 4 h later. We found that, while the PGE2-induced hyperalgesia was still significant at the 4th h in the vehicle-treated paws (white bars, t5=7.897, p=0.0005, when the mechanical nociceptive threshold at 4 h is compared to baseline; paired Student’s t-test), in the paws pretreated with MβC it was significantly attenuated by the 4th h after injection (black bars, second bar on the left: t5=1.136, p=0.3075, when the mechanical nociceptive threshold at 4 h is compared to baseline; paired Student’s t-test). To evaluate if the effect caused by lipid raft disruption on the expression of hyperalgesic priming was permanent, we tested again with PGE2 one week later, a period of time that allowed the recovery from the effect of MβC and return of the mechanical thresholds to baseline values (white bars: t5=0.1832; p=0.8018; black bars: t5=0.2225; p=0.8327, when comparing the mechanical thresholds before the first test with PGE2 and 1 week later; paired Student’s t-test). We observed, in both groups, that the mechanical hyperalgesia induced by PGE2 was still significant 4 h after injection, indicating that the attenuation of hyperalgesic priming by disruption of the lipid raft by MβC is reversible (white bars: t5=18.23; p<0.0001; black bars: t5=42.60; p<0.0001, when comparing the mechanical nociceptive threshold before and 4 h after PGE2 injection, respectively; paired Student’s t-test); Panel B: Rats that received intradermal injection of ψεRACK (1 μg; not shown) on the dorsum of the hind paw 2 weeks previously were treated, at the same site, with vehicle (white bars) or MβC (1 μg, black bars). 15 min later, CPA (1 μg) was injected and, the mechanical hyperalgesia, evaluated 30 min and 4 h later. Differently from what was observed in Panel A, where the disruption of lipid rafts significantly attenuated the expression of hyperalgesic priming (i.e., prolongation of PGE2-induced hyperalgesia), MβC did not affect the hyperalgesia induced by CPA in the primed paws (F1,10=0.13; p=0.4848, when comparing with the vehicle-treated group, two-way repeated measures ANOVA followed by Bonferroni post-test, n = 6 paws per group). This suggests that the hyperalgesic effect of CPA in the primed paw is not dependent on plasma membrane lipid rafts.
Figure 3
Figure 3. Schematic summary of the proposed mechanism in the plasma membrane involved in the prolongation of the PGE2-induced hyperalgesia in the primed nociceptor
In the normal, non-primed nociceptor (LEFT SIDE), the function of EP receptors is dependent on integrin β1 , . Activation of the receptor by intradermal injection PGE2 stimulates adenyl cyclase and cAMP production, which will, in turn, activate protein kinase A (PKA), and induce a short-lasting mechanical hyperalgesia, no longer present by the 4th h after injection . In the non-primed nociceptor, activation of the A1 receptor by adenosine does not affect the mechanical nociceptive threshold. On the other hand, in the primed nociceptor (RIGHT SIDE), in addition to the initial hyperalgesia induced by the activation of the PGE2 receptor-AC-PKA signaling pathway, a delayed component responsible for the prolongation of the PGE2 hyperalgesia, is observed. This late component is produced by the activation of an autocrine mechanism in which there is transport of cAMP to the extracellular space and further conversion to adenosine , followed by activation of the Gi-coupled A1 adenosine receptor (A1), which activates PKCε, prolonging the hyperalgesia produced by PGE2 in the primed nociceptor. This change in the profile of PGE2 hyperalgesia observed in hyperalgesic priming is dependent on the integrin β1 and lipid raft, the compartment in the plasma membrane where the components of the autocrine mechanism responsible for the conversion of cAMP to adenosine are located.
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