Enhanced spinal neuronal responses as a mechanism for the increased nociceptive sensitivity of interleukin-4 deficient mice

doi:10.1016/j.expneurol.2015.06.011

Experimental Neurology

Volume 271, September 2015, Pages 198-204

https://doi.org/10.1016/j.expneurol.2015.06.011 Get rights and content

Highlights

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Young interleukin-4 (IL-4) knockout (ko) mice are mechanically hypersensitive.
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Mechanical hypersensitivity disappears with aging in IL-4 ko mice.
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Young IL-4 ko mice have higher neuronal responses than WT and old IL-4 ko mice.
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IL-4 ko seems associated with age-dependent reduction in sensory neural activity.

Abstract

Lack of the anti-inflammatory and analgesic cytokine interleukin-4 (IL-4) is associated with mechanical hypersensitivity in mice, and low systemic levels of IL-4 are associated with pain in humans. We investigated whether the firing properties of murine nociceptive neurons in the spinal dorsal horn are affected by IL-4 deficiency. Single unit recordings from lumbar dorsal horn wide-dynamic-range (WDR) neurons were performed in IL-4 knock out (ko) mice and wild type (WT) littermates (3, 9, and 22 months old). We measured neuronal responses to innocuous (1 g) and noxious (26 g) von Frey mechanical stimulation at the plantar hind paw. Additionally, we induced secondary hyperalgesia by intraplantar injection of capsaicin and recorded discharges before and 5 and 10 min after injection. Baseline activity, activity upon innocuous stimulation, and postdischarges after noxious stimulation were not different between genotypes and ages. Responses to the noxious von Frey filament in 3 (34.5, 26.6–41.1 Hz) and 9 month old (49.7, 21.7–108.2 Hz) IL-4 ko mice were greater than in WT littermates (3 months, 18.1, 16.3–27.2 Hz, n.s.; 9 months, 33.6, 10.4–69.7 Hz; p < 0.05). In contrast, 22 month IL-4 ko mice had lower discharges (22.4, 16.8–28.9 Hz) than 3 and 9 month IL-4 ko mice (p < 0.01 each) and age-matched WT littermates (36.6, 10.4–59.4 Hz; n.s.). This pattern was also found 5 and 10 min after capsaicin injection. An enhanced excitability in the first segment of the nociceptive pathway may contribute to the increased behavioral responsiveness to painful stimuli of young IL-4 ko mice.

Introduction

Neuro-immune interactions via immune cells, neurotrophic factors, microRNAs, chemokines, and cytokines play a major role in the induction and maintenance of neuropathic pain (Austin and Moalem-Taylor, 2010, Calvo et al., 2012, Kress et al., 2013). As shown in animal models, the balanced expression of pro- and anti-inflammatory cytokines that have algesic and analgesic effects seems to be the basis for a physiological pain homeostasis. Altered behavior has been observed in animal models of cytokine or immune component deficiency (Üçeyler et al., 2010a, Üçeyler and Sommer, 2008).

While the role of the pro-inflammatory cytokines in pain has been investigated extensively, research on anti-inflammatory cytokines is scarce. The most frequently assessed candidates are interleukin-4 (IL-4), IL-10, and IL-13, which have analgesic effects in neuropathic and inflammatory pain models (Cunha et al., 1999, Hao et al., 2006, Karam et al., 2011, Ledeboer et al., 2007, Milligan et al., 2005, Vale et al., 2003). IL-4 connects the immune and opioid systems by inducing μ and δ opioid receptor transcription (Börner et al., 2004b, Kraus et al., 2001), which makes this cytokine an even more interesting candidate for pain research.

In previous studies we and others reported a reduced systemic expression of IL-4 in patients with chronic pain syndromes (Alexander et al., 2007, Üçeyler et al., 2006, Üçeyler et al., 2007). Correspondingly, we found that genetically modified IL-4 knock out (ko) mice have enhanced nociceptive reflexes upon mechanical stimulation with von Frey filaments (Üçeyler et al., 2011). In the present study we investigated whether an increased responsiveness of nociceptive neurons in the spinal dorsal horn underlies this increased pain sensitivity of IL-4 deficient mice. Single unit recordings were made from wide-dynamic-range (WDR) neurons in the lumbar dorsal horn of IL-4 ko mice and wild type (WT) littermates, and their discharges upon innocuous and noxious mechanical stimulation of a hind paw were compared. Additionally, we investigated the presence and extent of capsaicin-induced secondary hyperalgesia in genotypes and age groups. We found that spinal nociceptive neuronal responses were greater in young IL-4 ko mice than in WT mice. This might represent one of the reasons for the enhanced nociception of IL-4 deficient mice and humans. Unexpectedly, we also found that in old IL-4 ko mice the spinal nociceptive neuronal responses were lower than in WT mice.

Section snippets

Mice

Electrophysiological experiments were carried out in 81 male IL-4 ko mice (age 3 months: n = 20; 9 months: n = 42; 22 months: n = 19) and 62 WT littermates of C57BL/6J background (3 months: n = 12, 9 months: n = 32; 22 months: n = 18). For behavioral tests we additionally investigated 21 IL-4 ko mice and 16 WT littermates at the ages of 3 (n = 5 each), 10 (n = 5 each), and 18 (n = 11 IL-4 ko, n = 6 WT) months. IL-4 ko mice breeder pairs on C57BL/6J background were purchased from Jackson Laboratories (Maine, USA). WT

Baseline discharges

At baseline, i.e., during the 15 s period before the stimulus applications were begun, neuronal discharges reached a median of 0.2, 0.4, and 0.1 Hz in IL-4 ko, and of 1.6, 0.7, and 0.7 Hz in WT mice, of the three age groups, respectively (3, 9, and 22 months) without differences between genotypes or between age groups (Table 1).

Responses to mechanical stimulation

WDR neuronal responses to the 1 g von Frey filament reached a median of 3.6, 3.4, and 2.4 Hz for IL-4 ko, and 3.2, 1.3, and 1.1 Hz for WT mice of the three age groups,

Discussion

Systemic IL-4 deficiency is associated with chronic pain syndromes (Alexander et al., 2007, Üçeyler et al., 2006, Üçeyler et al., 2007) and IL-4 deficient mice display mechanical hypersensitivity upon stimulation with von Frey filaments (Üçeyler et al., 2011). We here report a spinal WDR neuronal responsiveness in adult IL-4 ko mice upon noxious mechanical stimulation of their receptive field that is greater than in their WT littermates. These results fit well with our previous findings (

Funding sources

The study was supported by intramural research funds of the University of Würzburg. CS and NÜ received funding from the European Union's Seventh Framework Programme (“ncRNAPain”, grant agreement number 602133). HV received travel grants from the German Humboldt-Stiftung and the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG (SO 328/6-1)). This work was part of the doctoral theses of SL and PS. SL received a six-month scholarship from the DFG (SFB 581).

Disclosures

SL, PS, and HV have no disclosures.

CG received honoraria for consultancy from Teva, and honoraria for presentations or travel grants from Biogen Idec, Allergan, Novartis, Merck Serono, Teva, and CSL Behring.

CS received honoraria for educational talks from the following pharmaceutical companies: Allergan, Astellas, Baxter, CSL Behring, Genzyme, Grifols, Kedrion, and Pfizer. Honoraria was received for consultancy from Baxter and Genzyme and research support from Genzyme.

NÜ received honoraria for