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. 2013 Oct;154(10):2207-2215.
doi: 10.1016/j.pain.2013.07.004. Epub 2013 Jul 4.

Electrophysiological correlates of hyperalgesic priming in vitro and in vivo

Jan Hendrich  1 , Pedro Alvarez, Elizabeth K Joseph, Xiaojie Chen, Oliver Bogen, Jon D Levine
Affiliations

Electrophysiological correlates of hyperalgesic priming in vitro and in vivo

Jan Hendrich et al. Pain. 2013 Oct.

Abstract

We have modeled the transition from acute to chronic pain in the rat. In this model (termed hyperalgesic priming) a chronic state develops after a prior inflammatory process or exposure to an inflammatory mediator, in which response to subsequent exposure to prostaglandin E2 (PGE2) is characterized by a protein kinase Cε-dependent marked prolongation of mechanical hyperalgesia. To assess the effect of priming on the function of the nociceptor, we have performed in vitro patch clamp and in vivo single-fiber electrophysiology studies using tumor necrosis factor α to induce priming. In vitro, the only change observed in nociceptors cultured from primed animals was a marked hyperpolarization in resting membrane potential (RMP); prolonged sensitization, measured at 60 minutes, could not be tested in vitro. However, complimentary with behavioral findings, in vivo baseline mechanical nociceptive threshold was significantly elevated compared to controls. Thirty minutes after injection of PGE2 into the peripheral receptive field, both primed and control nociceptors showed enhanced response to mechanical stimulation. However, 60 minutes after PGE2 administration, the response to mechanical stimulation was further increased in primed but not in control nociceptors. Thus, at the level of the primary afferent nociceptor, it is possible to demonstrate both altered function at baseline and prolonged PGE2-induced sensitization. Intrathecal antisense (AS) to Kv7.2, which contributes to RMP in sensory neurons, reversibly prevented the expression of priming in both behavioral and single-fiber electrophysiology experiments, implicating these channels in the expression of hyperalgesic priming.

Keywords: Action potential; Hyperalgesia; KCNQ; Nociceptor; Pain; Prostaglandin E(2); Resting membrane potential; Tumor necrosis factor alpha.

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Figures

Figure 1
Figure 1
Injection of TNFα into the gastrocnemius muscle produces mechanical hyperalgesia and hyperalgesic priming. (A) Single dose of rat recombinant TNFα (0.1-100 ng) injected into the gastrocnemius muscle of different groups of rats decreased the mechanical nociceptive threshold in a dose-dependent manner, in readings taken 2 hrs after its injection (n=6 per group). (B) The time-response curve for hyperalgesia induced by different doses of TNFα (n=6 per group). Muscle mechanical hyperalgesia reached a maximum 2 hrs post-injection (100 ng), and lasted up to 48 hrs. (C) Single doses of PGE2 (1-100 ng) injected into the gastrocnemius muscle produced a dose-dependent decrease in mechanical nociceptive in readings taken 30 min after injection (n=6 per group). (D) Mechanical hyperalgesia induced by different doses of PGE2 was investigated from 30 min to 24 hrs (n=6 per group). Hyperalgesia reached a maximum 30 min post-injection and lasted less than 4 hrs. (E) Previous injection of either 10 or 100 ng of PGE2, three days prior, did not change the amplitude or duration of mechanical hyperalgesia induced by a re-injection of 100 ng (5 μl) of PGE2 in the same injection point (n=6 per group). (F) Rats previously injected with TNFα exhibited prolonged duration of PGE2 (100 ng)-induced mechanical hyperalgesia compared to naïve rats (n=6). ***P < 0.001.
Figure 2
Figure 2
Action potential characteristics from primed DRG neurons in vitro. Representative traces (A) from naive (n=23) and primed (n=15) neurons show a significant hyperpolarization of resting membrane potential (RMP) in primed neurons (A, B), which occurs in the absence of any significant difference in other characteristics, for example membrane threshold of action potential generation (C). ***P<0.001
Figure 3
Figure 3
(A). Knockdown of the KCNQ2 expression in cultured dorsal root ganglia. Cultured DRGs treated with 1μM antisense ODN for 60 h in the presence of oligofectamine™ demonstrate a 27.5 ±1% decrease over all amplification cycles in their mRNA expression compared to cultured DRGs treated with 1 μM mismatch ODN. Column 1- DNA ladder, column 2- mismatch ODN, column 3- antisense ODN. (B) Application of Kv7.2 antisense prevents the expression of priming. Kv7.2 antisense (n=10) or mismatch (n=6) injections (10 μg intrathecal) were given daily for three days. On day four, nociceptive testing was performed in the antisense condition. The TNFα priming stimulus was applied (100 ng) followed by three more days of antisense / mismatch. On day seven, testing was performed before, 30 min and 4 h after PGE2 (100 ng) injection. Antisense treatment prevented the reduction in threshold observed 4 h after PGE2 injection. Nociceptive testing was performed again on Day 10 in antisense-treated rats (three days after cessation of treatment), the prolonged hyperalgesia 4 h after PGE2 injection had returned.
Figure 4
Figure 4
A measurement of the excitability of naive and primed nociceptive fibers in vivo. (A) C-fibers in primed neurons (n=35) displayed a significantly elevated mechanical threshold compared to naive fibers (n=44), that was reversed by antisense (n=10), but a similar conduction velocity (B). (C) Upon exposure to 100 ng 35 PGE2, mechanical threshold was not significantly altered in either primed (n=21) or Kv7.2 antisense-treated (n=10) fibers compared to naive fibers (n=10) after 60 min. (D) Compared to control fibers (n=10), the spike frequency at 60 min was increased in primed fibers (n=21) and decreased in primed fibers treated with antisense (n=10). (E) Spike response histogram of primed fibers measured before 100 ng PGE2 application, as well as 30 and 60 min after. *P<0.05, **P<0.01
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