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Peripheral nerve injury causes neuropathic pain including mechanical allodynia and thermal hyperalgesia due to central and peripheral sensitization. Spontaneous ectopic discharges derived from dorsal root ganglion (DRG) neurons and from the sites of injury are a key factor in the initiation of this sensitization. Numerous studies have focused primarily on DRG neurons; however, the injured axons themselves likely play an equally important role. Previous studies of neuropathic pain rats with spinal nerve ligation (SNL) showed that the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel in DRG neuronal bodies is important for the development of neuropathic pain. Here, we investigate the role of the axonal HCN channel in neuropathic pain rats. Using the chronic constriction injury (CCI) model, we found abundant axonal accumulation of HCN channel protein at the injured sites accompanied by a slight decrease in DRG neuronal bodies. The function of these accumulated channels was verified by local application of ZD7288, a specific HCN blocker, which significantly suppressed the ectopic discharges from injured nerve fibers with no effect on impulse conduction. Moreover, mechanical allodynia, but not thermal hyperalgesia, was relieved significantly by ZD7288. These results suggest that axonal HCN channel accumulation plays an important role in ectopic discharges from injured spinal nerves and contributes to the development of mechanical allodynia in neuropathic pain rats.

The large, medium-sized, and small neurons of the dorsal root ganglion (DRG) have different functions in the processing of various senses. Hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) contribute greatly to neuronal excitability. In the present study, which used whole-cell patch clamp techniques and immunohistochemical staining methods, the electrophysiological properties of DRG neurons were systematically compared, and the roles of HCN-1, -2, and -4 were examined. The main results were as follows. 1) The large neurons had significantly higher V0.5 values (membrane potential at which the HCN channels were half-activated) and shorter time constants (tau) than small or medium-sized DRG neurons. However, large DRG neurons had higher Ih density (HCN neuron current). 2) HCN-1 was found predominantly, but not exclusively, in large and medium-sized DRG neurons; HCN-2 was found in all DRG neurons; and HCN-4 was poorly visualized in all DRG neurons. HCN-1 and HCN-2 were colocalized in large and medium-sized neurons with immunostaining of adjacent sections. In the dorsal horn of the spinal cord, HCN-1, HCN-2, and HCN-4 were all expressed in laminae I-IV, although HCN-1 was not detectable in lamina II. 3) Blockade of Ih current in DRG neurons caused a significant decrease in V0.5, resting membrane potential, and repetitive firing number of action potential and a significant increase in time of rising phase of action potential. These results suggest that the different HCN channels in the three types of DRG neurons might contribute to their differential electrophysiological properties.

Although arterial limb tourniquet is one of the first-line treatments to prevent exsanguinating hemorrhage in both civilian pre-hospital and battlefield casualty care, prolonged application of a limb tourniquet can lead to serious ischemia-reperfusion injury. However, the underlying pathomechanisms of tourniquet-induced ischemia-reperfusion injury are still poorly understood. Using a murine model of acute limb ischemia-reperfusion, we investigated if acute limb ischemia-reperfusion injury is mediated by superoxide overproduction and mitochondrial dysfunction. Hind limbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Approximately 40% gastrocnemius muscle suffered infarction in this model. Activities of mitochondrial electron transport chain complexes including complex I, II, III, and IV in gastrocnemius muscle were decreased in the ischemia-reperfusion group compared to sham. Superoxide production was increased while activity of manganese superoxide dismutase (MnSOD, the mitochondria-targeted SOD isoform) was decreased in the ischemia-reperfusion group compared to sham group. Pretreatment with tempol (a SOD mimetic, 50 mg/kg) or co-enzyme Q10 (50 mg/kg) not only decreased the superoxide production, but also reduced the infarct size and normalized mitochondrial dysfunction in the gastrocnemius muscle. Our results suggest that tourniquet-induced skeletal muscle ischemia-reperfusion injuries including infarct size and mitochondrial dysfunction may be mediated via the superoxide over-production and reduced antioxidant activity. In the future, this murine ischemia-reperfusion model can be adapted to mechanistically evaluate anti-ischemic molecules in tourniquet-induced skeletal muscle injury.

Voltage-gated sodium (Nav) channels are responsible for initiation and propagation of action potential in the neurons. To explore the mechanisms for chronic heart failure (CHF)-induced baroreflex dysfunction, we measured the expression and current density of Nav channel subunits (Nav1.7, Nav1.8, and Nav1.9) in the aortic baroreceptor neurons and investigated the role of Nav channels on aortic baroreceptor neuron excitability and baroreflex sensitivity in sham and CHF rats. CHF was induced by left coronary artery ligation. The development of CHF (6–8 weeks after the coronary ligation) was confirmed by hemodynamic and morphological characteristics. Immunofluorescent data indicated that Nav1.7 was expressed in A-type (myelinated) and C-type (unmyelinated) nodose neurons but Nav1.8 and Nav1.9 were expressed only in C-type nodose neurons. Real-time RT-PCR and western blot data showed that CHF reduced mRNA and protein expression levels of Nav channels in nodose neurons. In addition, using the whole cell patch-clamp technique, we found that Nav current density and cell excitability of the aortic baroreceptor neurons were lower in CHF rats than that in sham rats. Aortic baroreflex sensitivity was blunted in anesthetized CHF rats, compared with that in sham rats. Furthermore, Nav channel activator (rATX II, 100 nM) significantly enhanced Nav current density and cell excitability of aortic baroreceptor neurons and improved aortic baroreflex sensitivity in CHF rats. These results suggest that reduced expression and activation of the Nav channels is involved in the attenuation of baroreceptor neuron excitability, which subsequently contributes to the impairment of baroreflex in CHF state.

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Chronic heart failure (CHF) is characterized by decreased cardiac parasympathetic and increased cardiac sympathetic nerve activity. This autonomic imbalance increases the risk of arrhythmias and sudden death in patients with CHF. We hypothesized that the molecular and cellular alterations of cardiac postganglionic parasympathetic (CPP) neurons located in the intracardiac ganglia and sympathetic (CPS) neurons located in the stellate ganglia (SG) possibly link to the cardiac autonomic imbalance in CHF. Rat CHF was induced by left coronary artery ligation. Single-cell real-time PCR and immunofluorescent data showed that L (Ca(v)1.2 and Ca(v)1.3), P/Q (Ca(v)2.1), N (Ca(v)2.2), and R (Ca(v)2.3) types of Ca2+ channels were expressed in CPP and CPS neurons, but CHF decreased the mRNA and protein expression of only the N-type Ca2+ channels in CPP neurons, and it did not affect mRNA and protein expression of all Ca2+ channel subtypes in the CPS neurons. Patch-clamp recording confirmed that CHF reduced N-type Ca2+ currents and cell excitability in the CPP neurons and enhanced N-type Ca2+ currents and cell excitability in the CPS neurons. N-type Ca2+ channel blocker (1 μM ω-conotoxin GVIA) lowered Ca2+ currents and cell excitability in the CPP and CPS neurons from sham-operated and CHF rats. These results suggest that CHF reduces the N-type Ca2+ channel currents and cell excitability in the CPP neurons and enhances the N-type Ca2+ currents and cell excitability in the CPS neurons, which may contribute to the cardiac autonomic imbalance in CHF.