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Intra-thalamus injection of ZD7288 reduced nociceptive behavior in rats
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Intra-thalamus injection of ZD7288 reduced nociceptive behavior in rats

Introduction: Chronic pain resulting from nerve injury or inflammation often exhibits mechanical allodynia and thermal hyperalgesia, which can persist for a prolonged period of time after the original injury. Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels are widely expressed in both peripheral sensory neurons and neurons in the central nervous system. The thalamus is the brain structure that receives projections from multiple ascending pain pathways and relays nociceptive input to the cortex and the limbic system. Human imaging study suggests thalamic dysfunction in neuropathic pain [1]. However, the molecular basis underlying the chronic pain mediated thalamic dysfunction in ascending pain pathway remains unclear. Considerable evidence indicates that dysfunction of Hyperpolarization-activated cyclic nucleotide-gated (HCN)  channel activity is associated with the development and maintenance of chronic pain and inhibition of HCN channel activity produces anti-nociceptive effect [2]. We hypothesized that increased HCN activity in the thalamus contributes to the chronic pain. We found that HCN immuno-reactivity is increased in the thalamus of rats with chronic pain and inhibition of HCN activity ameliorates mechanical allodynia and thermal hyperalgesia in rats with neuropathic pain or monoarthritis.

Method: Adult male Sprague–Dawley rats weighing 250-270 g were anesthetized with pentobarbital (50 mg/kg, i.p.). Chronic constriction injury (CCI) to sciatic nerve was performed. Briefly, one side the rat’s sciatic nerve was exposed in the mid-thigh, four ligatures using 4.0 mm chronic gut sutures were loosely placed around the sciatic nerve with a 1.0-1.5 mm interval between each ligature. Skin incision was closed with wound clips. Rats in sham groups underwent the same procedure but without nerve ligation. Complete Freud’s adjuvant (CFA)  or saline was injected into one side of the ankle articular cavity using a 28 gauge needle. A guide cannula (C315G with an infusion cannula C315I) was implanted in the ventral posterolateral (VPL) nucleus of the thalamus contralateral to the injury or CFA injection side. Saline and drug solutions were infused into the thalamus using a microinjection unit (33 gauge cannula) that extended 0.5 mm beyond the tip of the guide cannula. Mechanical allodynia: A von Frey filament was perpendicularly applied to the plantar surface of each hindpaw. A threshold force of response (in grams) was defined as the first filament that evoked at least two withdrawals out of  five application. Thermal hyperalgesia: Response to radiant heat was assessed using the foot-withdrawal test. Each animal underwent three trials and their results were averaged to yield mean withdrawal latencies. The cutoff was set at 20 seconds to avoid tissue damage. Immediately after the observation of behavior, Immunostaining and Western blot were carried out using the following primary and secondary antibodies: mouse anti-HCN1 antibody (1:800), mouse anti-HCN2 antibody (1:1000), Cy3- or FITC conjugated goat anti-mouse antibody (1:300), and HRP-conjugated  donkey anti-mouse antibody (1:10,000)  Primary antibody omission was used as a control. Sections were examined and captured with an Olympus fluorescence microscope. Western blot was analyzed using ImageJ. 

Result: Rats subjected to unilateral CCI exhibited mechanical allodynia and thermal hyperalgesia in the ipsilateral hindpaw as compared to sham controls (Fig. 1A and B). Both 1 µg and 5 µg of ZD7288 infusion attenuated mechanical allodynia and the effect of 5 µg of ZD7288 lasted for 90 min (Fig 1C and E). Thermal hyperalgesia was also reduced after the ZD7288 infusion in CCI rats and the higher ZD7288 dose (5 µg) did not further enhance the effect (Fig 1D and F). ZD7288 infusion also produced a prolonged anti-nociceptive effect on the hindpaw ipsilateral to CFA injection. By fourteen days after CCI and CFA, protein expression and immuno-reactivity of both HCN1 and HCN2 were increased in the contralateral thalamus as compared with that of sham rats analyzed by Western blot (Fig. 2A) and immunohistochemistry (Fig. 2B).

Discussion: Accumulating evidence suggests that abnormal HCN channel activity contributes to the development and maintenance of chronic pain. But the role of the thalamic HCN channel activity in chronic pain condition remains unknown. Our data suggest that HCN channel activity in the thalamus contributes to the behavioral manifestation of chronic pain in two rodent models without changing thermal or mechanical nociceptive threshold in sham rats. The VPL nucleus is a major relay site of the spinothalamic track for pain and temperature sensation. In rats with neuropathic pain, neurons in the VPL nucleus exhibited higher spontaneous firing as well as evoked response. Moreover, increased HCN channel activity may contribute to ectopic firing under chronic pain. Our data show that inhibition of HCN channel activity in the VPL nucleus alleviated chronic pain. It has been shown that intrathecal or intra-periaquaductal gray application of HCN channel inhibitor reduced neuropathic pain behaviors. These data lend strong support for a critical role of the brain HCN channel activity in chronic pain. our data show that inhibition of HCN channel activity in the VPL nucleus alleviated chronic pain.

Conclusion: We report that infusion of a HCN blocker ZD7288 into the VPL nucleus of the thalamus attenuated nociceptive behavior in rats with chronic pain induced by peripheral nerve injury or inflammation. We also observed that HCN1 and HCN2 protein expression was increased in the thalamus of the same rats after nerve injury. Among four HCN channel subunits (HCN1-4), HCN1 and HCN2 subunits are expressed in neurons of both peripheral and central nerve systems and are highly relevant to pain processing and modulation, the current data suggest that inhibition of HCN subtype channel activity may be a method to alleviate chronic pain. Due to bradycardia resulting from blocking the sinoatrial HCN4 with all known Ih blockers, broad HCN channel blockers may not be useful when applied systemically. The present results suggest that searching for HCN subtype blockers relevant for pain would be beneficial to patients with chronic pain.

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