Frequency-Dependent Neural Modulation of Dorsal Horn Neurons by Kilohertz Spinal Cord Stimulation in Rats.

Kilohertz high-frequency spinal cord stimulation (kHF-SCS) is a rapidly advancing neuromodulatory technique in the clinical management of chronic pain. However, the precise cellular mechanisms underlying kHF-SCS-induced paresthesia-free pain relief, as well as the neural responses within spinal pain circuits, remain largely unexplored. In this study, using a novel preparation, we investigated the impact of varying kilohertz frequency SCS on dorsal horn neuron activation. Employing calcium imaging on isolated spinal cord slices, we found that extracellular electric fields at kilohertz frequencies (1, 3, 5, 8, and 10 kHz) induce distinct patterns of activation in dorsal horn neurons. Notably, as the frequency of extracellular electric fields increased, there was a clear and significant monotonic escalation in neuronal activity. This phenomenon was observed not only in superficial dorsal horn neurons, but also in those located deeper within the dorsal horn. Our study demonstrates the unique patterns of dorsal horn neuron activation in response to varying kilohertz frequencies of extracellular electric fields, and we contribute to a deeper understanding of how kHF-SCS induces paresthesia-free pain relief. Furthermore, our study highlights the potential for kHF-SCS to modulate sensory information processing within spinal pain circuits. These insights pave the way for future research aimed at optimizing kHF-SCS parameters and refining its therapeutic applications in the clinical management of chronic pain.

Simultaneous 10 kHz and 40 Hz spinal cord stimulation increases dorsal horn inhibitory interneuron activity.

Since 1967, spinal cord stimulation (SCS) has been used to manage chronic intractable pain of the trunk and limbs. Low-intensity, paresthesia-free, 10 kHz SCS has demonstrated statistically- and clinically-superior long-term pain relief compared to conventional SCS. 10 kHz SCS has been proposed to operate via selective activation of inhibitory interneurons in the superficial dorsal horn. In contrast, 40 Hz SCS is presumed to operate largely via dorsal column fiber activation. To determine if these mechanisms may be implemented synergistically, we examined the effect of each type of stimulation both independently and simultaneously on putatively inhibitory and putatively excitatory neurons in the superficial dorsal horn. When 10 kHz SCS was applied relatively caudally to the measured spinal segment, simultaneous with 40 Hz SCS applied relatively rostrally to that spinal segment, inhibitory interneurons demonstrated a median increase of 26 spikes/s compared to their baseline firing rates. Median firing rate increases of inhibitory interneurons were 8.7 and 5.1 spikes/s during 40 Hz SCS applied rostrally and 10 kHz SCS applied caudally, respectively. By comparison, the median firing rate of excitatory interneurons increased by 4.1 spikes/s during simultaneous 40 Hz SCS applied rostrally and 10 kHz SCS applied caudally. Median firing rate increases of excitatory interneurons were 13 and 0.8 spikes/s during 40 Hz SCS applied rostrally and 10 kHz SCS applied caudally, respectively. This suggests that simultaneously applying 10 kHz SCS caudally and 40 Hz SCS rostrally may provide greater pain relief than either type of SCS alone by increasing the firing rates of inhibitory interneurons, albeit with greater excitatory interneuron activation.

Low-Intensity 10 kHz Spinal Cord Stimulation Reduces Behavioral and Neural Hypersensitivity in a Rat Model of Painful Diabetic Neuropathy.

Background: Low-intensity 10 kHz spinal cord stimulation (SCS) has been shown to provide pain relief in patients with chronic pain resulting from diabetic peripheral neuropathy (DPN). However to date, there have been no studies of 10 kHz SCS in animal models of diabetes. We aimed to establish correlative data of the effects of this therapy on behavioral and electrophysiological measures in a DPN model. Methods: Twenty-five adult male Sprague-Dawley rats were injected once intraperitoneally with 60 mg/kg streptozotocin (STZ) to induce diabetes over a subsequent 4 w period, while 4 naïve control animals were not injected. After approximately 21 d, 12 of STZ-injected rats had mini epidural SCS leads implanted: 8 received continuous low intensity (~30% motor threshold) 10 kHz SCS, and 4 received sham SCS (0 mA) over 7 d. Behavioral assays (von Frey filament probe of hindpaw) were measured in 18 animals and in vivo dorsal horn electrophysiological studies (receptive field; response to afferent brush, von Frey probe, pinch) were performed in 17 animals. Results: Across behavioral assays of mechanical allodynia and electrophysiological assays of receptive field size and mechanosensitivity, diabetic animals stimulated with 10 kHz SCS showed statistically significant improvements compared to sham SCS. Conclusion: Low-intensity 10 kHz SCS produced several measures associated with a reduction of pain in diabetic rodent models that may help explain the clinical benefits of 10 kHz SCS in patients with painful diabetic neuropathy.

Differential Modulation of Dorsal Horn Neurons by Various Spinal Cord Stimulation Strategies.

New strategies for spinal cord stimulation (SCS) for chronic pain have emerged in recent years, which may work better via different analgesic mechanisms than traditional low-frequency (e.g., 50 Hz) paresthesia-based SCS. To determine if 10 kHz and burst SCS waveforms might have a similar mechanistic basis, we examined whether these SCS strategies at intensities ostensibly below sensory thresholds would modulate spinal dorsal horn (DH) neuronal function in a neuron type-dependent manner. By using an in vivo electrophysiological approach in rodents, we found that low-intensity 10 kHz SCS, but not burst SCS, selectively activates inhibitory interneurons in the spinal DH. This study suggests that low-intensity 10 kHz SCS may inhibit pain-sensory processing in the spinal DH by activating inhibitory interneurons without activating DC fibers, resulting in paresthesia-free pain relief, whereas burst SCS likely operates via other mechanisms.

Low-intensity, Kilohertz Frequency Spinal Cord Stimulation Differently Affects Excitatory and Inhibitory Neurons in the Rodent Superficial Dorsal Horn.

Since 1967, spinal cord stimulation (SCS) has been used to manage chronic intractable pain of the trunk and limbs. Compared to traditional high-intensity, low-frequency (<100 Hz) SCS that is thought to produce paresthesia and pain relief by stimulating large myelinated fibers in the dorsal column (DC), low-intensity, high-frequency (10 kHz) SCS has demonstrated long-term pain relief without generation of paresthesia. To understand this paresthesia-free analgesic mechanism of 10 kHz SCS, we examined whether 10 kHz SCS at intensities below sensory thresholds would modulate spinal dorsal horn (DH) neuronal function in a neuron type-dependent manner. By using in vivo and ex vivo electrophysiological approaches, we found that low-intensity (sub-sensory threshold) 10 kHz SCS, but not 1 kHz or 5 kHz SCS, selectively activates inhibitory interneurons in the spinal DH. This study suggests that low-intensity 10 kHz SCS may inhibit pain sensory processing in the spinal DH by activating inhibitory interneurons without activating DC fibers, resulting in paresthesia-free pain relief.

Excitatory neurons are more disinhibited than inhibitory neurons by chloride dysregulation in the spinal dorsal horn.

Neuropathic pain is a debilitating condition caused by the abnormal processing of somatosensory input. Synaptic inhibition in the spinal dorsal horn plays a key role in that processing. Mechanical allodynia - the misperception of light touch as painful - occurs when inhibition is compromised. Disinhibition is due primarily to chloride dysregulation caused by hypofunction of the potassium-chloride co-transporter KCC2. Here we show, in rats, that excitatory neurons are disproportionately affected. This is not because chloride is differentially dysregulated in excitatory and inhibitory neurons, but, rather, because excitatory neurons rely more heavily on inhibition to counterbalance strong excitation. Receptive fields in both cell types have a center-surround organization but disinhibition unmasks more excitatory input to excitatory neurons. Differences in intrinsic excitability also affect how chloride dysregulation affects spiking. These results deepen understanding of how excitation and inhibition are normally balanced in the spinal dorsal horn, and how their imbalance disrupts somatosensory processing.

Chloride Dysregulation through Downregulation of KCC2 Mediates Neuropathic Pain in Both Sexes.

The behavioral features of neuropathic pain are not sexually dimorphic despite sex differences in the underlying neuroimmune signaling. This raises questions about whether neural processing is comparably altered. Here, we test whether the K+-Cl- co-transporter KCC2, which regulates synaptic inhibition, plays an equally important role in development of neuropathic pain in male and female rodents. Past studies on KCC2 tested only males. We find that inhibiting KCC2 in uninjured animals reproduces behavioral and electrophysiological features of neuropathic pain in both sexes and, consistent with equivalent injury-induced downregulation of KCC2, that counteracting chloride dysregulation reverses injury-induced behavioral and electrophysiological changes in both sexes. These findings demonstrate that KCC2 downregulation contributes equally to pain hypersensitivity in males and females. Whereas diverse (and sexually dimorphic) mechanisms regulate KCC2, regulation of intracellular chloride relies almost exclusively on KCC2. Directly targeting KCC2 thus remains a promising strategy for treatment of neuropathic pain in both sexes.

Microglial pannexin-1 channel activation is a spinal determinant of joint pain.

Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, yet effective therapies are lacking. We identify the pannexin-1 (Panx1) channel as a therapeutic target for alleviating mechanical allodynia, a cardinal sign of arthritis. In rats, joint pain caused by intra-articular injection of monosodium iodoacetate (MIA) was associated with spinal adenosine 5'-triphosphate (ATP) release and a microglia-specific up-regulation of P2X7 receptors (P2X7Rs). Blockade of P2X7R or ablation of spinal microglia prevented and reversed mechanical allodynia. P2X7Rs drive Panx1 channel activation, and in rats with mechanical allodynia, Panx1 function was increased in spinal microglia. Specifically, microglial Panx1-mediated release of the proinflammatory cytokine interleukin-1ß (IL-1ß) induced mechanical allodynia in the MIA-injected hindlimb. Intrathecal administration of the Panx1-blocking peptide 10panx suppressed the aberrant discharge of spinal laminae I-II neurons evoked by innocuous mechanical hindpaw stimulation in arthritic rats. Furthermore, mice with a microglia-specific genetic deletion of Panx1 were protected from developing mechanical allodynia. Treatment with probenecid, a clinically used broad-spectrum Panx1 blocker, resulted in a striking attenuation of MIA-induced mechanical allodynia and normalized responses in the dynamic weight-bearing test, without affecting acute nociception. Probenecid reversal of mechanical allodynia was also observed in rats 13 weeks after anterior cruciate ligament transection, a model of posttraumatic osteoarthritis. Thus, Panx1-targeted therapy is a new mechanistic approach for alleviating joint pain.

Chloride dysregulation and inhibitory receptor blockade yield equivalent disinhibition of spinal neurons yet are differentially reversed by carbonic anhydrase blockade.

Synaptic inhibition plays a key role in processing somatosensory information. Blocking inhibition at the spinal level is sufficient to produce mechanical allodynia, and many neuropathic pain conditions are associated with reduced inhibition. Disinhibition of spinal neurons can arise through decreased GABAA/glycine receptor activation or through dysregulation of intracellular chloride. We hypothesized that these distinct disinhibitory mechanisms, despite all causing allodynia, are differentially susceptible to therapeutic intervention. Specifically, we predicted that reducing bicarbonate efflux by blocking carbonic anhydrase with acetazolamide (ACTZ) would counteract disinhibition caused by chloride dysregulation without affecting normal inhibition or disinhibition caused by GABAA/glycine receptor blockade. To test this, responses to innocuous tactile stimulation were recorded in vivo from rat superficial dorsal horn neurons before and after different forms of pharmacological disinhibition and again after application of ACTZ. Blocking GABAA or glycine receptors caused hyperresponsiveness equivalent to that caused by blocking the potassium chloride cotransporter KCC2, but, consistent with our predictions, only disinhibition caused by KCC2 blockade was counteracted by ACTZ. ACTZ did not alter responses of neurons with intact inhibition. As pathological downregulation of KCC2 is triggered by brain-derived neurotrophic factor, we also confirmed that ACTZ was effective against brain-derived neurotrophic factor-induced hyperresponsiveness. Our results argue that intrathecal ACTZ has antiallodynic effects only if allodynia arises through chloride dysregulation; therefore, behavioral evidence that ACTZ is antiallodynic in nerve-injured animals affirms the contribution of chloride dysregulation as a key pathological mechanism. Although different disinhibitory mechanisms are not mutually exclusive, these results demonstrate that their relative contribution dictates which specific therapies will be effective.

Criticality and degeneracy in injury-induced changes in primary afferent excitability and the implications for neuropathic pain.

Neuropathic pain remains notoriously difficult to treat despite numerous drug targets. Here, we offer a novel explanation for this intractability. Computer simulations predicted that qualitative changes in primary afferent excitability linked to neuropathic pain arise through a switch in spike initiation dynamics when molecular pathologies reach a tipping point (criticality), and that this tipping point can be reached via several different molecular pathologies (degeneracy). We experimentally tested these predictions by pharmacologically blocking native conductances and/or electrophysiologically inserting virtual conductances. Multiple different manipulations successfully reproduced or reversed neuropathic changes in primary afferents from naïve or nerve-injured rats, respectively, thus confirming the predicted criticality and its degenerate basis. Degeneracy means that several different molecular pathologies are individually sufficient to cause hyperexcitability, and because several such pathologies co-occur after nerve injury, that no single pathology is uniquely necessary. Consequently, single-target-drugs can be circumvented by maladaptive plasticity in any one of several ion channels. DOI: http://dx.doi.org/10.7554/eLife.02370.001.

Inflammatory mediators potentiate high affinity GABA(A) currents in rat dorsal root ganglion neurons.

Following acute tissue injury action potentials may be initiated in afferent processes terminating in the dorsal horn of the spinal cord that are propagated back out to the periphery, a process referred to as a dorsal root reflex (DRR). The DRR is dependent on the activation of GABA(A) receptors. The prevailing hypothesis is that DRR is due to a depolarizing shift in the chloride equilibrium potential (E(Cl)) following an injury-induced activation of the Na(+)-K(+)-Cl(-)-cotransporter. Because inflammatory mediators (IM), such as prostaglandin E(2) are also released in the spinal cord following tissue injury, as well as evidence that E(Cl) is already depolarized in primary afferents, an alternative hypothesis is that an IM-induced increase in GABA(A) receptor mediated current (I(GABA)) could underlie the injury-induced increase in DRR. To test this hypothesis, we explored the impact of IM (prostaglandin E(2) (1 µM), bradykinin (10 µM), and histamine (1 µM)) on I(GABA) in dissociated rat dorsal root ganglion (DRG) neurons with standard whole cell patch clamp techniques. IM potentiated I(GABA) in a subpopulation of medium to large diameter capsaicin insensitive DRG neurons. This effect was dependent on the concentration of GABA, manifest only at low concentrations (<10 µM). THIP evoked current were also potentiated by IM and GABA (1 µM) induced tonic currents enhanced by IM were resistant to gabazine (20 µM). The present data are consistent with the hypothesis that an acute increase in I(GABA) contributes to the emergence of injury-induced DRR.

Inflammation-induced changes in BK(Ca) currents in cutaneous dorsal root ganglion neurons from the adult rat.

BACKGROUND: Inflammation-induced sensitization of primary afferents is associated with a decrease in K(+) current. However, the type of K(+) current and basis for the decrease varies as a function of target of innervation. Because glabrous skin of the rat hindpaw is used often to assess changes in nociception in models of persistent pain, the purpose of the present study was to determine the type and extent to which K(+) currents contribute to the inflammation-induced sensitization of cutaneous afferents. Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion neurons from naïve and inflamed (3 days post complete Freund's adjuvant injection) rats were studied with whole cell and perforated patch techniques. RESULTS: Inflammation-induced sensitization of small diameter cutaneous neurons was associated with an increase in action potential duration and rate of decay of the afterhyperpolarization. However, no changes in voltage-gated K(+) currents were detected. In contrast, Ca(2+) modulated iberiotoxin sensitive and paxilline sensitive K(+) (BK(Ca)) currents were significantly smaller in small diameter IB4+ neurons. This decrease in current was not associated with a detectable change in total protein levels of the BK(Ca) channel α or ß subunits. Single cell PCR analysis revealed a significant change in the pattern of expression of α subunit splice variants and ß subunits that were consistent, at least in part, with inflammation-induced changes in the biophysical properties of BK(Ca) currents in cutaneous neurons. CONCLUSIONS: Results of this study provide additional support for the conclusion that it may be possible, if not necessary to selectively treat pain arising from specific body regions. Because a decrease in BK(Ca) current appears to contribute to the inflammation-induced sensitization of cutaneous afferents, BK(Ca) channel openers may be effective for the treatment of inflammatory pain.

Mitochondrial Ca(2+) uptake is essential for synaptic plasticity in pain.

The increase of cytosolic free Ca(2+) ([Ca(2+)](c)) due to NMDA receptor activation is a key step for spinal cord synaptic plasticity by altering cellular signal transduction pathways. We focus on this plasticity as a cause of persistent pain. To provide a mechanism for these classic findings, we report that [Ca(2+)](c) does not trigger synaptic plasticity directly but must first enter into mitochondria. Interfering with mitochondrial Ca(2+) uptake during a [Ca(2+)](c) increase blocks induction of behavioral hyperalgesia and accompanying downstream cell signaling, with reduction of spinal long-term potentiation (LTP). Furthermore, reducing the accompanying mitochondrial superoxide levels lessens hyperalgesia and LTP induction. These results indicate that [Ca(2+)](c) requires downstream mitochondrial Ca(2+) uptake with consequent production of reactive oxygen species (ROS) for synaptic plasticity underlying chronic pain. These results suggest modifying mitochondrial Ca(2+) uptake and thus ROS as a type of chronic pain therapy that should also have broader biologic significance.

Reactive oxygen species contribute to neuropathic pain by reducing spinal GABA release.

Although both a loss of spinal inhibitory neurotransmission and the involvement of oxidative stress have been regarded as important mechanisms in the pathogenesis of pain, the relationship between these 2 mechanisms has not been studied. To determine whether reactive oxygen species (ROS) involvement in pain mechanisms is related to the diminished inhibitory transmission in the substantia gelatinosa (SG) of the spinal dorsal horn, behavioral studies and whole-cell recordings were performed in FVB/NJ mice. Neuropathic pain was induced by a tight ligation of the L5 spinal nerve (SNL). Pain behaviors in the affected foot were assessed by behavioral testing for mechanical hyperalgesia. Pain behaviors developed by 3 days and lasted more than 8 weeks. Both systemic and intrathecal administration of an ROS scavenger, phenyl-N-tert-butylnitrone (PBN), temporarily reversed mechanical hyperalgesia up to 2 hours, 1 week after SNL. In nonligated mice, an intrathecal injection of an ROS donor, tert-butyl hydroperoxide (t-BOOH), dose-dependently induced mechanical hyperalgesia for 1.5 hours. In whole-cell voltage clamp recordings of SG neurons, perfusion with t-BOOH significantly decreased the frequency of mIPSCs, and this effect was reversed by PBN. Furthermore, t-BOOH decreased the frequency of GABA(A) receptor-mediated mIPSCs without altering their amplitudes but did not affect glycine receptor-mediated mIPSCs. In SNL mice, mIPSC frequency in SG neurons was significantly reduced as compared with that of normal mice, which was restored by PBN. The antihyperalgesic effect of PBN on mechanical hyperalgesia was attenuated by intrathecal bicuculline, a GABA(A) receptor blocker. Our results indicate that the increased ROS in spinal cord may induce pain by reducing GABA inhibitory influence on SG neurons that are involved in pain transmission.

Involvement of reactive oxygen species in long-term potentiation in the spinal cord dorsal horn.

Recent studies suggest that reactive oxygen species (ROS) are functional messenger molecules in central sensitization, an underlying mechanism of persistent pain. Because spinal cord long-term potentiation (LTP) is the electrophysiological basis of central sensitization, this study investigates the effects of the increased or decreased spinal ROS levels on spinal cord LTP. Spinal cord LTP is induced by either brief, high-frequency stimulation (HFS) of a dorsal root at C-fiber intensity or superfusion of a ROS donor, tert-butyl hydroperoxide (t-BOOH), onto rat spinal cord slice preparations. Field excitatory postsynaptic potentials (fEPSPs) evoked by dorsal root stimulations with either Abeta- or C-fiber intensity are recorded from the superficial dorsal horn. HFS significantly increases the slope of both Abeta- and C-fiber evoked fEPSPs, thus suggesting LTP development. The induction, not the maintenance, of HFS-induced LTP is blocked by a N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosphonopentanoic acid (D-AP5). Both the induction and maintenance of LTP of Abeta-fiber-evoked fEPSPs are inhibited by a ROS scavenger, either N-tert-butyl-alpha-phenylnitrone or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl. A ROS donor, t-BOOH-induced LTP is inhibited by N-tert-butyl-alpha-phenylnitrone but not by D-AP5. Furthermore, HFS-induced LTP and t-BOOH-induced LTP occlude each other. The data suggest that elevated ROS is a downstream event of NMDA receptor activation and an essential step for potentiation of synaptic excitability in the spinal dorsal horn.