Geza Gemes

Species and strain differences in rodent sciatic nerve anatomy: Implications for studies of neuropathic pain

&NA; Hindlimb pain models developed in rats have been transposed to mice, but assumed sciatic nerve neuroanatomic similarities have not been examined. We compared sciatic nerve structural organization in mouse strains (C57BL/6J, DBA/2J, and B6129PF2/J) and rat strains (Wistar, Brown Norway, and Sprague–Dawley). Dissection and retrograde labeling showed mouse sciatic nerve origins predominantly from the third lumbar (L3) and L4 spinal nerves, unlike the L4 and L5 in rats. Proportionate contributions by each level differed significantly between strains in both mice and rats. Whereas all rats had six lumbar vertebrae, variable patterns in mice included mostly five vertebrae in DBA/2J, mostly six vertebrae in C57BL/6J, and a mix in B6129PF2/J. Mice with a short lumbar vertebral column showed a rostral shift in relative contributions to the sciatic nerve by L3 and L4. Ligation of the mouse L4 nerve created hyperalgesia similar to that in rats after L5 ligation, and motor changes were similar after mouse L4 and rat L5 ligation (foot cupping) and after mouse L3 and rat L4 ligation (flexion weakness). Thus, mouse L3 and L4 neural segments are anatomically and functionally homologous with rat L4 and L5 segments. Neuronal changes after distal injury or inflammation should be sought in the mouse L3 and L4 ganglia, and the spinal nerve ligation model in mice should involve ligation of the L4 nerve while L3 remains intact. Strain‐dependent variability in segmental contributions to the sciatic nerve may account in part for genetic differences in pain behavior after spinal nerve ligation.

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.

Store-Operated Ca2+ Entry in Sensory Neurons: Functional Role and the Effect of Painful Nerve Injury

Painful nerve injury disrupts levels of cytoplasmic and stored Ca2+ in sensory neurons. Since influx of Ca2+ may occur through store-operated Ca2+ entry (SOCE) as well as voltage- and ligand-activated pathways, we sought confirmation of SOCE in sensory neurons from adult rats and examined whether dysfunction of SOCE is a possible pathogenic mechanism. Dorsal root ganglion neurons displayed a fall in resting cytoplasmic Ca2+ concentration when bath Ca2+ was withdrawn, and a subsequent elevation of cytoplasmic Ca2+ concentration (40 ± 5 nm) when Ca2+ was reintroduced, which was amplified by store depletion with thapsigargin (1 μm), and was significantly reduced by blockers of SOCE, but was unaffected by antagonists of voltage-gated membrane Ca2+ channels. We identified the underlying inwardly rectifying Ca2+-dependent ICRAC (Ca2+ release activated current), as well as a large thapsigargin-sensitive inward current activated by withdrawal of bath divalent cations, representing SOCE. Molecular components of SOCE, specifically STIM1 and Orai1, were confirmed in sensory neurons at both the transcript and protein levels. Axonal injury by spinal nerve ligation (SNL) elevated SOCE and ICRAC. However, SOCE was comparable in injured and control neurons when stores were maximally depleted by thapsigargin, and STIM1 and Orai1 levels were not altered by SNL, showing that upregulation of SOCE after SNL is driven by store depletion. Blockade of SOCE increased neuronal excitability in control and injured neurons, whereas injured neurons showed particular dependence on SOCE for maintaining levels of cytoplasmic and stored Ca2+, which indicates a compensatory role for SOCE after injury.

Guidance of Block Needle Insertion by Electrical Nerve Stimulation: A Pilot Study of the Resulting Distribution of Injected Solution in Dogs

Background:Little is known regarding the final needle tip location when various intensities of nerve stimulation are used to guide block needle insertion. Therefore, in control and hyperglycemic dogs, the authors examined whether lower-intensity stimulation results in injection closer to the sciatic nerve than higher-threshold stimulation. Methods:During anesthesia, the sciatic nerve was approached with an insulated nerve block needle emitting either 1 mA (high-current group, n = 9) or 0.5 mA (low-current group, n = 9 in control dogs and n = 6 in hyperglycemic dogs). After positioning to obtain a distal motor response, the lowest current producing a response was identified, and ink (0.5 ml) was injected. Frozen sections of the tissue revealed whether the ink was in contact with the epineurium of the nerve, distant to it, or within it. Results:In control dogs, the patterns of distribution using high-threshold (final current 0.99 ± 0.03 mA, mean ± SD) and low-threshold (final current 0.33 ± 0.08 mA) stimulation equally showed ink that was in contact with the epineurium or distant to it. One needle placement in the high-threshold group resulted in intraneural injection. In hyperglycemic dogs, all needle insertions used a low-threshold technique (n = 6, final threshold 0.35 ± 0.08 mA), and all resulted in intraneural injections. Conclusions:In normal dogs, current stimulation levels in the range of 0.33–1.0 mA result in needle placement comparably close to the sciatic nerve but do not correlate with distance from the target nerve. In this experimental design, low-threshold electrical stimulation does not offer satisfactory protection against intraneural injection in the presence of hyperglycemia.

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.

Increasing arterial oxygen partial pressure during cardiopulmonary resuscitation is associated with improved rates of hospital admission

AIM As recent clinical data suggest a harmful effect of arterial hyperoxia on patients after resuscitation from cardiac arrest (CA), we aimed to investigate this association during cardiopulmonary resuscitation (CPR), the earliest and one of the most crucial phases of recirculation. METHODS We analysed 1015 patients who from 2003 to 2010 underwent out-of-hospital CPR administered by emergency medical services serving 300,000 inhabitants. Inclusion criteria for further analysis were nontraumatic background of CA and patients >18 years of age. One hundred and forty-five arterial blood gas analyses including oxygen partial pressure (paO2) measurement were obtained during CPR. RESULTS We observed a highly significant increase in hospital admission rates associated with increases in paO2 in steps of 100 mmHg (13.3 kPa). Subsequently, data were clustered according to previously described cutoffs (≤ 60 mmHg [8 kPa]], 61-300 mmHg [8.1-40 kPa], >300 mmHg [>40 kPa]). Baseline variables (age, sex, initial rhythm, rate of bystander CPR and collapse-to-CPR time) of the three compared groups did not differ significantly. Rates of hospital admission after CA were 18.8%, 50.6% and 83.3%, respectively. In a multivariate analysis, logistic regression revealed significant prognostic value for paO2 and the duration of CPR. CONCLUSION This study presents novel human data on the arterial paO2 during CPR in conjunction with the rate of hospital admission. We describe a significantly increased rate of hospital admission associated with increasing paO2. We found that the previously described potentially harmful effects of hyperoxia after return of spontaneous circulation were not reproduced for paO2 measured during CPR. CLINICAL TRIAL REGISTRATION n/a.

Learned Avoidance from noxious mechanical Simulation but not Threshold Semmes Weinstein filament Stimulation after Nerve Injury in Rats

UNLABELLED Noxious mechanical stimulation evokes a complex and sustained hyperalgesic motor response after peripheral nerve injury that contrasts with a brief and simple withdrawal seen after noxious stimulation in control animals or after threshold punctate mechanical stimulation by the von Frey technique. To test which of these behaviors indicate pain, the aversiveness of the experience associated with each was determined using a passive avoidance test in rats after sciatic nerve ligation (SNL) or skin incision alone. After 18 days, step-down latency was measured during 9 sequential trials at 10-minute intervals. At each trial, rats received either no stimulus, needle stimuli, or threshold Semmes Weinstein (SW) filament stimuli after stepping down. Reactions were either a hyperalgesic response or a brief reflexive withdrawal. In SNL animals, needle stimulation produced substantial learned avoidance when animals showed hyperalgesic responses but produced minimal prolonged latency in SNL animals that showed only simple withdrawal responses. No learned avoidance developed using threshold SW testing in SNL animals. These findings show that needle stimulation is aversive in rats responding with hyperalgesic behavior. In contrast, SW stimulation, as well as needle stimulation that produced mere withdrawal, is minimally aversive. PERSPECTIVE The validity of measures of pain in animals is open to question. We demonstrated that needle stimulation is aversive in rats that respond with hyperalgesic-type behavior and is therefore a valid indicator of pain. Stimulation by SW is minimally aversive and is a problematic indicator of pain.

KATP channel subunits in rat dorsal root ganglia: alterations by painful axotomy

BackgroundATP-sensitive potassium (KATP) channels in neurons mediate neuroprotection, they regulate membrane excitability, and they control neurotransmitter release. Because loss of DRG neuronal KATP currents is involved in the pathophysiology of pain after peripheral nerve injury, we characterized the distribution of the KATP channel subunits in rat DRG, and determined their alterations by painful axotomy using RT-PCR, immunohistochemistry and electron microscopy.ResultsPCR demonstrated Kir6.1, Kir6.2, SUR1 and SUR2 transcripts in control DRG neurons. Protein expression for all but Kir6.1 was confirmed by Western blots and immunohistochemistry. Immunostaining of these subunits was identified by fluorescent and confocal microscopy in plasmalemmal and nuclear membranes, in the cytosol, along the peripheral fibers, and in satellite glial cells. Kir6.2 co-localized with SUR1 subunits. Kir6.2, SUR1, and SUR2 subunits were identified in neuronal subpopulations, categorized by positive or negative NF200 or CGRP staining. KATP current recorded in excised patches was blocked by glybenclamide, but preincubation with antibody against SUR1 abolished this blocking effect of glybenclamide, confirming that the antibody targets the SUR1 protein in the neuronal plasmalemmal membrane.In the myelinated nerve fibers we observed anti-SUR1 immunostaining in regularly spaced funneled-shaped structures. These structures were identified by electron microscopy as Schmidt-Lanterman incisures (SLI) formed by the Schwann cells. Immunostaining against SUR1 and Kir6.2 colocalized with anti-Caspr at paranodal sites.DRG excised from rats made hyperalgesic by spinal nerve ligation exhibited similar staining against Kir6.2, SUR1 or SUR2 as DRG from controls, but showed decreased prevalence of SUR1 immunofluorescent NF200 positive neurons. In DRG and dorsal roots proximal to axotomy SLI were smaller and showed decreased SUR1 immunofluorescence.ConclusionsWe identified Kir6.2/SUR1 and Kir6.2/SUR2 KATP channels in rat DRG neuronal somata, peripheral nerve fibers, and glial satellite and Schwann cells, in both normal state and after painful nerve injury. This is the first report of KATP channels in paranodal sites adjacent to nodes of Ranvier and in the SLI of the Schwann cells. After painful axotomy KATP channels are downregulated in large, myelinated somata and also in SLI, which are also of smaller size compared to controls.Because KATP channels may have diverse functional roles in neurons and glia, further studies are needed to explore the potential of KATP channels as targets of therapies against neuropathic pain and neurodegeneration.

ATP-sensitive potassium currents in rat primary afferent neurons: biophysical, pharmacological properties, and alterations by painful nerve injury

ATP-sensitive potassium (K(ATP)) channels may be linked to mechanisms of pain after nerve injury, but remain under-investigated in primary afferents so far. We therefore characterized these channels in dorsal root ganglion (DRG) neurons, and tested whether they contribute to hyperalgesia after spinal nerve ligation (SNL). We compared K(ATP) channel properties between DRG somata classified by diameter into small or large, and by injury status into neurons from rats that either did or did not become hyperalgesic after SNL, or neurons from control animals. In cell-attached patches, we recorded basal K(ATP) channel opening in all neuronal subpopulations. However, higher open probabilities and longer open times were observed in large compared to small neurons. Following SNL, this channel activity was suppressed only in large neurons from hyperalgesic rats, but not from animals that did not develop hyperalgesia. In contrast, no alterations of channel activity developed in small neurons after axotomy. On the other hand, cell-free recordings showed similar ATP sensitivity, inward rectification and unitary conductance (70-80 pS) between neurons classified by size or injury status. Likewise, pharmacological sensitivity to the K(ATP) channel opener diazoxide, and to the selective blockers glibenclamide and tolbutamide, did not differ between groups. In large neurons, selective inhibition of whole-cell ATP-sensitive potassium channel current (I(K(ATP))) by glibenclamide depolarized resting membrane potential (RMP). The contribution of this current to RMP was also attenuated after painful axotomy. Using specific antibodies, we identified SUR1, SUR2, and Kir6.2 but not Kir6.1 subunits in DRGs. These findings indicate that functional K(ATP) channels are present in normal DRG neurons, wherein they regulate RMP. Alterations of these channels may be involved in the pathogenesis of neuropathic pain following peripheral nerve injury. Their biophysical and pharmacological properties are preserved even after axotomy, suggesting that K(ATP) channels in primary afferents remain available for therapeutic targeting against established neuropathic pain.

Axotomy Depletes Intracellular Calcium Stores in Primary Sensory Neurons

Background:The cellular mechanisms of neuropathic pain are inadequately understood. Previous investigations have revealed disrupted Ca2+ signaling in primary sensory neurons after injury. The authors examined the effect of injury on intracellular Ca2+ stores of the endoplasmic reticulum, which critically regulate the Ca2+ signal and neuronal function. Methods:Intracellular Ca2+ levels were measured with Fura-2 or mag-Fura-2 microfluorometry in axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent L4 neurons isolated from hyperalgesic rats after L5 spinal nerve ligation, compared to neurons from control animals. Results:Endoplasmic reticulum Ca2+ stores released by the ryanodine-receptor agonist caffeine decreased by 46% in axotomized small neurons. This effect persisted in Ca2+-free bath solution, which removes the contribution of store-operated membrane Ca2+ channels, and after blockade of the mitochondrial, sarco-endoplasmic Ca2+-ATPase and the plasma membrane Ca2+ ATPase pathways. Ca2+ released by the sarco-endoplasmic Ca2+-ATPase blocker thapsigargin and by the Ca2+-ionophore ionomycin was also diminished by 25% and 41%, respectively. In contrast to control neurons, Ca2+ stores in axotomized neurons were not expanded by neuronal activation by K+ depolarization, and the proportionate rate of refilling by sarco-endoplasmic Ca2+-ATPase was normal. Luminal Ca2+ concentration was also reduced by 38% in axotomized neurons in permeabilized neurons. The adjacent neurons of the L4 dorsal root ganglia showed modest and inconsistent changes after L5 spinal nerve ligation. Conclusions:Painful nerve injury leads to diminished releasable endoplasmic reticulum Ca2+ stores and a reduced luminal Ca2+ concentration. Depletion of Ca2+ stores may contribute to the pathogenesis of neuropathic pain.