Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, PC34
Chronic inflammation increases expression of hyperpolarization-activated and cyclic nucleotide-gated channel subunit 2 in small dorsal root ganglion neurons in the rat
T. M. Smith1, L. Djouhri1
1. University of Liverpool, Liverpool, United Kingdom.
Chronic inflammatory pain is characterised by hypersensitivity to painful stimuli (hyperalgesia) and/or normally non-painful stimuli (allodynia). This hypersensitivity is partly due to sensitization of primary afferent dorsal root ganglion (DRG) neurons innervating inflamed tissue. These sensitised neurons exhibit increased excitability (Woolf & Ma, 2007). Hyperpolarization-activated and cyclic nucleotide-gated cation channels, which consist of four subunits (HCN1-4), are key modulators of neuronal activity (Biel et al. 2009). The aim of the present study was to examine whether chronic inflammation alters HCN1-3 channel protein expression in DRG neurons. We focused on these subunits because they are clearly expressed in rat DRG neurons, whereas expression of HCN4 is uncertain (Chaplan et al. 2003). Inflammation was induced by two injections of 100µl of Complete Freund’s adjuvant (CFA) into the left hindpaw and left knee region of female Wistar rats (150-175g) under 3% isoflurane anaesthesia. Seven says post CFA, we performed immunohistochemistry in L4/L5 DRGs and spinal cord, from control and CFA treated rats, using well characterised anti-HCN1-3 antibodies (Alomone Laboratories). We also examined both the control and CFA tissues for co-localization of HCN1-3 with isolectin IB4, a neuronal marker of a subpopulation of C-fibre neurons. The somatic sizes of HCN1-3 positive neurons were measured and divided into 3 groups: small diameter (≤30 µm), medium-diameter (~31-40 µm) and large diameter (>40 µm) neurons.Seven days after CFA-induced inflammation, there was a significant increase (P < 0.01, Mann-Whitney U test) in HCN2-immunoreactivity in small neurons (putative nociceptors) determined by densitometric comparison of DRG sections from control and CFA rats and in the percentage of small and medium DRG neurons-expressing HCN2 (P<0.01, Fisher's exact test). Increased HCN2-immunoreactivity was also seen in axons (possibly both the peripheral and central processes) of these small neurons-expressing HCN2, as well as the superficial dorsal horn of the spinal cord, where the central terminals of C- and Aδ- nociceptive neurons terminate. In contrast, there was no change in HCN1- or HCN3- immunoreactivity in any cell-type of DRG neurons or spinal cord following CFA-induced inflammation. These results are consistent with recent findings (Papp et al. 2010) that show increased HCN2-immunoreactivity in the axon terminals of C- and Aδ-fibre neurons in the superficial dorsal of the spinal cord, 3 days after CFA-induced inflammation. Taken altogether, the data suggest that HCN2 channels contribute to the hyperexcitability of DRG neurons and hypersensitivity associated with tissue inflammation.
Where applicable, experiments conform with Society ethical requirements