J. Bruce McCallum

Detection of Neuropathic Pain in a Rat Model of Peripheral Nerve Injury

Background:Behavioral criteria that confirm neuropathic pain in animal injury models are undefined. Therefore, the authors sought clinically relevant measures that distinguish pain behavior of rats with peripheral nerve injury from those with sham injury. Methods:The authors examined mechanical and thermal sensory sensitivity, comparing responses at baseline to responses after spinal nerve ligation (SNL group), sham nerve injury (sham group), or skin incision alone (control group). Results:Substantial variance was evident in all sensory tests at baseline. After surgery, tests using brush, cold, or heat stimulation showed minimal distinctions between surgical groups. Postsurgical thresholds for flexion withdrawal from mechanical stimulation with von Frey fibers were decreased bilaterally in SNL and sham groups. In contrast, the probability of a complex hyperalgesia-type response with prolonged elevation, shaking, or licking of the paw was selectively increased on the ipsilateral side in the SNL group. Nonetheless, the effect of SNL on behavior was inconsistent, regardless of the sensory test. The behavioral measure that best distinguishes between SNL and sham groups and thereby best identifies animals with successful SNL-induced neuropathic pain is increased ipsilateral postsurgical probability of a hyperalgesia-type response to noxious mechanical stimulation. Using receiver operating characteristics analysis, mechanical hyperalgesia identifies a local SNL effect in approximately 60% of animals when specificity is required to be 90% or higher. Conclusions:Simple withdrawal from von Frey tactile stimulation, although frequently used, is not a valid measure of peripheral nerve injury pain in rats, whereas a complex hyperalgesic-type response is a specific neuropathy-induced behavior.

Distinct membrane effects of spinal nerve ligation on injured and adjacent dorsal root ganglion neurons in rats.

Background:Painful peripheral nerve injury results in disordered sensory neuron function that contributes to the pathogenesis of neuropathic pain. However, the relative roles of neurons with transected axons versus intact adjacent neurons have not been resolved. An essential first step is identification of electrophysiologic changes in these two neuronal populations after partial nerve damage. Methods:Twenty days after spinal nerve ligation (SNL), intracellular recordings were obtained from axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent, intact L4 neurons, as well as from control neurons and others subjected to sham-SNL surgery. Results:Pronounced electrophysiologic changes were seen only in L5 neurons after SNL. Both Aα/β and A&dgr; neuron types showed increased action potential duration, decreased afterhyperpolarization amplitude and duration, and decreased current threshold for action potential initiation. Aα/β neurons showed resting membrane potential depolarization, and increased repetitive firing during sustained depolarization developed in A&dgr; neurons. The afterhyperpolarization duration in neurons with C fibers shortened after axotomy. In contrast to the axotomized L5 neurons, neighboring L4 neurons showed no changes in action potential duration, afterhyperpolarization dimensions, or excitability after SNL. Depolarization rate (dV/dt) increased after SNL in L4 Aα/β and A&dgr; neurons but decreased in L5 neurons. Time-dependent rectification during hyperpolarizing current injection (sag) was greater after SNL in Aα/β L4 neurons compared with L5. Sham-SNL surgery produced only a decreased input resistance in Aα/β neurons and a decreased conduction velocity in medium-sized cells. In the L5 ganglion after axotomy, a novel set of neurons, consisting of 24% of the myelinated population, exhibited long action potential durations despite myelinated neuron conduction velocities, particularly depolarized resting membrane potential, low depolarization rate, and absence of sag. Conclusions:These findings indicate that nerve injury–induced electrical instability is restricted to axotomized neurons and is absent in adjacent intact neurons.

Painful neuropathy decreases membrane calcium current in mammalian primary afferent neurons

&NA; Hyperexcitability of the primary afferent neuron leads to neuropathic pain following injury to peripheral axons. Changes in calcium channel function of sensory neurons following injury have not been directly examined at the channel level, even though calcium is a primary second messenger‐regulating neuronal function. We compared calcium currents (ICa) in 101 acutely isolated dorsal root ganglion neurons from 31 rats with neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve, to cells from 25 rats with normal sensory function following sham surgery. Cells projecting to the sciatic nerve were identified with a fluorescent label applied at the CCI site. Membrane function was determined using patch‐clamp techniques in current clamp mode, and in voltage‐clamp mode using solutions and conditions designed to isolate ICa. Somata of peripheral sensory neurons from hyperalgesic rats demonstrated decreased ICa. Peak calcium channel current density was diminished by injury from 3.06±0.30 pS/pF to 2.22±0.26 pS/pF in medium neurons, and from 3.93±0.38 pS/pF to 2.99±0.40 pS/pF in large neurons. Under these voltage and pharmacologic conditions, medium‐sized neuropathic cells lacked obvious T‐type calcium currents which were present in 25% of medium‐sized cells from control animals. Altered Ca2+ signalling in injured sensory neurons may contribute to hyperexcitability leading to neuropathic pain.

Nitric oxide activates ATP-sensitive potassium channels in mammalian sensory neurons: action by direct S-nitrosylation

BackgroundATP-sensitive potassium (KATP) channels in neurons regulate excitability, neurotransmitter release and mediate protection from cell-death. Furthermore, activation of KATP channels is suppressed in DRG neurons after painful-like nerve injury. NO-dependent mechanisms modulate both KATP channels and participate in the pathophysiology and pharmacology of neuropathic pain. Therefore, we investigated NO modulation of KATP channels in control and axotomized DRG neurons.ResultsCell-attached and cell-free recordings of KATP currents in large DRG neurons from control rats (sham surgery, SS) revealed activation of KATP channels by NO exogenously released by the NO donor SNAP, through decreased sensitivity to [ATP]i.This NO-induced KATP channel activation was not altered in ganglia from animals that demonstrated sustained hyperalgesia-type response to nociceptive stimulation following spinal nerve ligation. However, baseline opening of KATP channels and their activation induced by metabolic inhibition was suppressed by axotomy. Failure to block the NO-mediated amplification of KATP currents with specific inhibitors of sGC and PKG indicated that the classical sGC/cGMP/PKG signaling pathway was not involved in the activation by SNAP. NO-induced activation of KATP channels remained intact in cell-free patches, was reversed by DTT, a thiol-reducing agent, and prevented by NEM, a thiol-alkylating agent. Other findings indicated that the mechanisms by which NO activates KATP channels involve direct S-nitrosylation of cysteine residues in the SUR1 subunit. Specifically, current through recombinant wild-type SUR1/Kir6.2 channels expressed in COS7 cells was activated by NO, but channels formed only from truncated isoform Kir6.2 subunits without SUR1 subunits were insensitive to NO. Further, mutagenesis of SUR1 indicated that NO-induced KATP channel activation involves interaction of NO with residues in the NBD1 of the SUR1 subunit.ConclusionNO activates KATP channels in large DRG neurons via direct S-nitrosylation of cysteine residues in the SUR1 subunit. The capacity of NO to activate KATP channels via this mechanism remains intact even after spinal nerve ligation, thus providing opportunities for selective pharmacological enhancement of KATP current even after decrease of this current by painful-like nerve injury.

Loss of T-type calcium current in sensory neurons of rats with neuropathic pain.

Background Pathophysiology in the primary sensory neuron may contribute to chronic neuropathic pain. Ca channels play a central role in neuronal processes, and sensory neurons are rich in low-voltage–activated calcium channels (LVACCs). However, the physiologic function of these channels is unknown. Their possible role in rebound burst firing makes them a candidate for increased excitability after neuropathic injury. Methods This study uses pharmacological methods to isolate LVACC in cells from the dorsal root ganglia of neuropathic and sham-operated rats, including the blockade of high-voltage–activated Ca channels with fluoride and selective toxins. LVACCs were examined with conventional whole cell patch clamp electrophysiology techniques. Results After chronic constriction injury of the peripheral axon, LVACC was significantly reduced compared to sham rats as shown by a 60% reduction in peak current density and an 80% reduction in total calcium influx. A depolarizing shift in the voltage dependence of activation and an increase in the rate of deactivation and inactivation appear to cause this reduction of LVACC. Either Ni2+ or mibefradil, blockers of LVACC, applied in the bath to normal dorsal root ganglion cells during current clamp significantly and reversibly increased excitability. Conclusions These results suggest that loss of LVACC may contribute to decreased spike frequency adaptation and increased excitability after injury to sensory neurons. Through decreased Ca2+ influx, the cell becomes less stable and more likely to initiate or transmit bursts of action potentials. Consequently, modulation of Ca2+ currents at the dorsal root ganglion may be a potential method of therapeutic intervention.

Painful Peripheral Nerve Injury Decreases Calcium Current in Axotomized Sensory Neurons

Background:Reports of Ca2+ current (ICa) loss after injury to peripheral sensory neurons do not discriminate between axotomized and spared neurons. The spinal nerve ligation model separates axotomized from spared neurons innervating the same site. The authors hypothesized that ICa loss is a result of neuronal injury, so they compared axotomized L5 dorsal root ganglion neurons to spared L4 neurons, as well as neurons from rats undergoing skin incision alone. Methods:After behavioral testing, dissociated neurons from L4 and L5 dorsal root ganglia were studied in both current and voltage patch clamp modes. The biophysical consequence of ICa loss on the action potential was confirmed using selective ICa antagonists. Data were grouped into small, medium, and large cells for comparison. Results:Reduced ICa was predominantly a consequence of axotomy (L5 after spinal nerve ligation) and was most evident in small and medium neurons. ICa losses were associated with action potential prolongation in small and medium cells, whereas the amplitude and duration of after hyperpolarization was reduced in medium and large neurons. Blockade with Ca2+ channel antagonists showed that action potential prolongation and after hyperpolarization diminution were alike, attributable to the loss of ICa. Conclusion:Axotomy is required for ICa loss. ICa loss correlated with changes in the biophysical properties of sensory neuron membranes during action potential generation, which were due to ICa loss leading to decreased outward Ca2+-sensitive K+ currents. Taken together, these results suggest that neuropathic pain may be mediated, in part, by loss of ICa and the cellular processes dependent on Ca2+.

Suppressed Ca2+/CaM/CaMKII-dependent KATP channel activity in primary afferent neurons mediates hyperalgesia after axotomy

Painful axotomy decreases KATP channel current (IKATP) in primary afferent neurons. Because cytosolic Ca2+ signaling is depressed in injured dorsal root ganglia (DRG) neurons, we investigated whether Ca2+–calmodulin (CaM)–Ca2+/CaM-dependent kinase II (CaMKII) regulates IKATP in large DRG neurons. Immunohistochemistry identified the presence of KATP channel subunits SUR1, SUR2, and Kir6.2 but not Kir6.1, and pCaMKII in neurofilament 200–positive DRG somata. Single-channel recordings from cell-attached patches revealed that basal and evoked IKATP by ionomycin, a Ca2+ ionophore, is activated by CaMKII. In axotomized neurons from rats made hyperalgesic by spinal nerve ligation (SNL), basal KATP channel activity was decreased, and sensitivity to ionomycin was abolished. Basal and Ca2+-evoked KATP channel activity correlated inversely with the degree of hyperalgesia induced by SNL in the rats from which the neurons were isolated. Inhibition of IKATP by glybenclamide, a selective KATP channel inhibitor, depolarized resting membrane potential (RMP) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry. The selective KATP channel opener diazoxide hyperpolarized the RMP and attenuated neurotransmitter release. Axotomized neurons from rats made hyperalgesic by SNL lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide (AIPm), a pseudosubstrate blocker of CaMKII, whereas axotomized neurons from SNL animals that failed to develop hyperalgesia showed normal IKATP inhibition by AIPm. AIPm also depolarized RMP in control neurons via KATP channel inhibition. Unitary current conductance and sensitivity of KATP channels to cytosolic ATP and ligands were preserved even after painful nerve injury, thus providing opportunities for selective therapeutic targeting against neuropathic pain.

Opposing effects of spinal nerve ligation on calcium-activated potassium currents in axotomized and adjacent mammalian primary afferent neurons

UNLABELLED Calcium-activated potassium channels regulate AHP and excitability in neurons. Since we have previously shown that axotomy decreases I(Ca) in DRG neurons, we investigated the association between I(Ca) and K((Ca)) currents in control medium-sized (30-39 microM) neurons, as well as axotomized L5 or adjacent L4 DRG neurons from hyperalgesic rats following L5 SNL. Currents in response to AP waveform voltage commands were recorded first in Tyrodes solution and sequentially after: 1) blocking Na(+) current with NMDG and TTX; 2) addition of K((Ca)) blockers with a combination of apamin 1 microM, iberiotoxin 200 nM, and clotrimazole 500 nM; 3) blocking remaining K(+) current with the addition of 4-AP, TEA-Cl, and glibenclamide; and 4) blocking I(Ca) with cadmium. In separate experiments, currents were evoked (HP -60 mV, 200 ms square command pulses from -100 to +50 mV) while ensuring high levels of activation of I(K(Ca)) by clamping cytosolic Ca(2+) concentration with pipette solution in which Ca(2+) was buffered to 1 microM. This revealed I(K(Ca)) with components sensitive to apamin, clotrimazole and iberiotoxin. SNL decreases total I(K(Ca)) in axotomized (L5) neurons, but increases total I(K(Ca)) in adjacent (L4) DRG neurons. All I(K(Ca)) subtypes are decreased by axotomy, but iberiotoxin-sensitive and clotrimazole-sensitive current densities are increased in adjacent L4 neurons after SNL. In an additional set of experiments we found that small-sized control DRG neurons also expressed iberiotoxin-sensitive currents, which are reduced in both axotomized (L5) and adjacent (L4) neurons. CONCLUSIONS Axotomy decreases I(K(Ca)) due to a direct effect on K((Ca)) channels. Axotomy-induced loss of I(Ca) may further potentiate current reduction. This reduction in I(K(Ca)) may contribute to elevated excitability after axotomy. Adjacent neurons (L4 after SNL) exhibit increased I(K(Ca)) current.

Norepinephrine infusion into nucleus basalis elicits microarousal in desflurane-anesthetized rats

Background: The nucleus basalis of Meynert of the basal forebrain has been implicated in the regulation of the state of consciousness across normal sleep-wake cycles. Its role in the modulation of general anesthesia was investigated. Methods: Rats were chronically implanted with bilateral infusion cannulae in the nucleus basalis of Meynert and epidural electrodes to record the electroencephalogram in frontal and visual cortices. Animals were anesthetized with desflurane at a concentration required for the loss of righting reflex (4.6 ± 0.5%). Norepinephrine (17.8 nmol) or artificial cerebrospinal fluid was infused at 0.2 &mgr;l/min (1 &mgr;l total). Behavioral response to infusion was measured by scoring the orofacial, limb, and head movements, and postural changes. Results: Behavioral responses were higher after norepinephrine (2.1 ± 1) than artificial cerebrospinal fluid (0.63 ± 0.8) infusion (P < 0.01, Student t test). Responses were brief (1–2 min), repetitive, and more frequent after norepinephrine infusion (P < 0.0001, chi-square test). Electroencephalogram delta power decreased after norepinephrine in frontal (70 ± 7%) but not in visual cortex (P < 0.05, Student t test). Simultaneously, electroencephalogram cross-approximate entropy between frontal and visual cortices increased from 3.17 ± 0.56 to 3.85 ± 0.29 after norepinephrine infusion (P < 0.01, Student t test). Behavioral activation was predictable by the decrease in frontal delta power (logistic regression, P < 0.05). Conclusions: Norepinephrine infusion into the nucleus basalis of Meynert can modulate anesthetic depth presumably by ascending activation of the cortex. The transient nature of the responses suggests a similarity with microarousals normally observed during natural sleep, and may imply a mechanism for transient awareness under light anesthesia.

Ca2+-Dependent Regulation of Ca2+ Currents in Rat Primary Afferent Neurons: Role of CaMKII and the Effect of Injury

Currents through voltage-gated Ca2+ channels (ICa) may be regulated by cytoplasmic Ca2+ levels ([Ca2+]c), producing Ca2+-dependent inactivation (CDI) or facilitation (CDF). Since ICa regulates sensory neuron excitability, altered CDI or CDF could contribute to pain generation after peripheral nerve injury. We explored this by manipulating [Ca2+]c while recording ICa in rat sensory neurons. In uninjured neurons, elevating [Ca2+]c with a conditioning prepulse (−15 mV, 2 s) inactivated ICa measured during subsequent test pulses (−15 mV, 5 ms). This inactivation was Ca2+-dependent (CDI), since it was decreased with elimination of Ca2+ influx by depolarization to above the ICa reversal potential, with high intracellular Ca2+ buffering (EGTA 10 mm or BAPTA 20 mm), and with substitution of Ba2+ for extracellular Ca2+, revealing a residual voltage-dependent inactivation. At longer latencies after conditioning (>6 s), ICa recovered beyond baseline. This facilitation also proved to be Ca2+-dependent (CDF) using the protocols limiting cytoplasmic Ca2+ elevation. Ca2+/calmodulin-dependent protein kinase II (CaMKII) blockers applied by bath (KN-93, myristoyl-AIP) or expressed selectively in the sensory neurons (AIP) reduced CDF, unlike their inactive analogues. Protein kinase C inhibition (chelerythrine) had no effect. Selective blockade of N-type Ca2+ channels eliminated CDF, whereas L-type channel blockade had no effect. Following nerve injury, CDI was unaffected, but CDF was eliminated in axotomized neurons. Excitability of sensory neurons in intact ganglia from control animals was diminished after a similar conditioning pulse, but this regulation was eliminated by injury. These findings indicate that ICa in sensory neurons is subject to both CDI and CDF, and that hyperexcitability following injury-induced loss of CDF may result from diminished CaMKII activity.