Deleting HCN2 ion channel isoform carried by nociceptor afferents makes mice pain-free after inflammatory damage.

Researchers suggest that blocking a subset of HCN2 ion channels in pain signaling nociceptor neurons may provide a new approach to combating both inflammatory and neuropathic pain. Their research in mice showed that genetic depletion of the voltage gated sodium channel Na1.8 in nociceptive afferent neurons effectively blocked the repetitive firing responsible for trigger pain sensation. Interestingly, deleting the channel had no effect on the animals’ normal acute pain responses, only their responses to neuropathic stimuli.

The team, from the U.K.’s University of Cambridge and the University of Cádiz in Spain, say their findings implicated repetitive firing specifically in neurons expressing the voltage-dependent sodium channel isoform NaV1.8 as the originating event in neuropathic pain. Reporting in Science, Peter A. McNaughton, Ph.D., and colleagues conclude that “HCN2-selective blockers may have value as analgesics to combat both inflammatory and neuropathic pain.” Their paper is titled “HCN2 Ion Channels Play a Central Role in Inflammatory and Neuropathic Pain.”

The team engineered two strains of mice. One was globally deficient in HCN2 (HCN2–/– ), and the other was specifically deficient in the NaV1.8, which is expressed only in nociceptive primary sensory neurons. Compared with wild-type animals, the NaV1.8-HCN2–/–  mice were phenotypically normal, had similar heat thresholds and performance on the rotarod test of motor function, as well as showed similar paw swelling in response to prostaglandin E2 (PGE2) injection.

Electrophysiological studies on individual neurons showed that knocking out HCN2 in small sensory neurons—the major of which are nociceptors—affected neuronal response to elevated levels of forskolin (FSK) or PGE2-induced cAMP and essentially blocked action potential firing. An increase in cAMP caused a depolarizing shift in the voltage dependence of activation of Ih in wild-type neurons but not in global HCN2 –/– neurons.  The sensitivity to FSK of the firing rate in HCN2 –/– neurons could be restored by transfection of HCN2.

Small neurons taken from NaV1.8-HCN2 –/– mice could be classified as either wild-type, in which the voltage activation curve shifted positive after an increase in cAMP, and “HCN2–/–”, in which there was no effect of cAMP on voltage activation.

“The effects of HCN2 deletion on action potential firing in small sensory neurons suggest that HCN2 may be important in inflammatory pain, where the firing of action potentials in response to a noxious stimulus is enhanced by inflammatory mediators such as PGE2,” the researchers note. To investigate this further they tested the effects of injecting formalin into the hind paws of wild-type and NaV1.8-HCN2–/– animals. Formalin injection normally causes two phases of pain behavior, an initial phase caused by direct activation of nociceptors, and a later phase caused by release of inflammatory mediators, the authors note.

They found that the pre-injection pain thresholds of NaV1.8-HCN2–/–mice to both heat and mechanical stimuli were indistinguishable from those of wild-type mice. However, after formalin injection, NaV1.8-HCN2–/– animals demonstrated about a 50% reduction in late phase pain effects, when compared with wild-type animals.

Because formalin actually engages multiple pain and nonpain pathways, the researchers then moved on to evaluate the effects of PGE2 injection, which induces more specific stimuli. PGE2-induced thermal hyperalgesia was abolished in NaV1.8-HCN2–/–mice and also in wild-type animals administered with the nonspecific HCN-blocker ZD7288. Thermal hyperalgesia was also completely abolished in NaV1.8-HCN2–/– mice following administration of carrageenan, which causes more longlasting inflammation, and also in wild-type mice treated with ZD7288. In contrast, mechanical hyperalgesia was unaffected either by NaV1.8-specific HCN2 deletion or by ZD7288 therapy in wild-type mice.

Neuropathic pain is a long-lasting pain state that in animal models is associated with maintained firing in C-fibres, which suggests a possible involvement of repetitive activity driven by HCN2, the authors continue. To investigate this further the team examined the effect of NaV1.8-specific HCN2 deletion using the chronic constriction injury (CCI) neuropathic pain model. They tested for thermal hyperalgesia, mechanical hyperalgesia, and cold allodynia, which are all substantially enhanced in neuropathic pain.

What they found was that while animals expressing normal levels of HCN2 demonstrated prominent and prolonged hyperalgesia to thermal and mechanical stimuli, in NaV1.8-HCN2–/– mice both heat and mechanical thresholds were indistinguishable from sham-operated animals. Cold allodynia, tested by placing the mice on a cold plate and comparing the number of paw lifts ipsilateral and contralateral to the nerve lesion, was also absent only in NaV1.8-HCN2–/– animals.

“Our results show that removal of HCN2 in the NaV1.8-expressing subpopulation of small afferent neurons abolishes the effects of PGE2, both in enhancing action potential firing in isolated neurons and in producing inflammatory thermal hyperalgesia in vivo,” the authors write.  In particular, they stress, PGE2 is well established to be the downstream target of NSAID analgesics, and the reported results show that HCN2 is an important target of PGE2.

The finding that deleting HCN2 from NaV1.8-expressing sensory neurons completely abolished neuropathic pain—even though HCN2 was not deleted in all small neurons—suggested that only the NaV1.8-expressing population is critical for initiating neuropathic pain, they continue. “We propose that an inflammatory mediator, released at the site of nerve injury, acts to modify the voltage dependence of activation of HCN2 in nociceptive afferents expressing NaV1.8, thus promoting repetitive firing of action potentials and initiating neuropathic pain.”

The fact that targeting a specific ion channel affects only neuropathic-type pain is particularly important, professor NcNaughton stresses. “Many genes play a critical role in pain sensation, but in most cases interfering with them simply abolishes all pain,or even all sensation. What is exciting about the work on the HCN2 gene is that removing it—or blocking it pharmacologically—eliminates neuropathic pain without affecting normal acute pain. This finding could be very valuable clinically because normal pain sensation is essential for avoiding accidental damage.”

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