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This review considers the ion channels that underlie transduction of nociceptive energies in the periphery, that are involved in impulse initiation and propagation in peripheral sensory neurones, and that participate in pre- and post-synaptic actions in the spinal cord dorsal horn, in light of their susceptibility to local anaesthetics. Although there are results from experiments on isolated cells and tissues ex vivo that support the hypothesized actions, their extrapolation to actions in vivo and the consequences for peri- and postoperative pain control are largely speculative. [ABSTRACT FROM AUTHOR]

Oxytocin has known antinociceptive effects and is upregulated perinatally. This pilot study investigated the association of plasma oxytocin and postcesarean incisional pain. Plasma samples from 18 patients undergoing elective cesarean delivery were drawn at 1 hour preoperatively and 1 and 24 hours postoperatively and analyzed by using enzyme-linked immunosorbent assay. Pain was assessed at 1 day, 8 weeks, 3 months, and 6 months postoperatively. Incisional pain at 24 hours was inversely correlated with 1- and 24-hour oxytocin levels, with higher plasma oxytocin associated with lower pain (ρ, -0.52 and -0.66; P < .05).

Background and Objectives: Local anesthetics (LAs) are often infiltrated subcutaneously for localized perioperative numbing. In addition to blocking nerve conduction, LAs act on pathways used by a variety of pain-inducing and inflammatory mediators. We describe the effects in isolated model sensory neurons of LAs on responses to the algogenic and sensitizing peptide, bradykinin (BK)., Methods: ND/7 sensory neurons were stimulated by different concentrations of BK in the presence or absence of LAs, with transient increases in intracellular calcium (Δ[Ca]in) detected fluorometrically in fields of cells. Equilibrium saturable binding of radiolabeled BK also was conducted on the same type of cells, with and without LA., Results: Responses to low BK (5 nM) were inhibited by lidocaine at 1 mM (approximately 35% inhibition) and 10 mM (approximately 70% inhibition), whereas responses to high BK (100 nM) were unaffected by 1 mM yet inhibited (approximately 75%) by 10 mM lidocaine. Bupivacaine (1 and 2 mM) did not reduce peak Δ[Ca]in (using 5 nM BK). Lidocaine's quaternary derivative, QX-314 (10 mM), also was ineffective on peak Ca (5 nM BK). Saturation binding of BK showed that lidocaine lowered the binding capacity (Bmax) without changing the KD, consistent with noncompetitive inhibition., Conclusions: At subclinical concentrations, lidocaine suppresses BK's activation of model sensory neurons. This effect adds to the known analgesic mechanisms of LAs and likely contributes to the reduction of postincisional pain.

Thoracotomy results in chronic postoperative pain (CPTP) in half of the cases. Earlier findings in rat models of persistent post-surgical pain suggest that spinal pathways are critical for pain onset but not its maintenance. Descending systems from the brain stem modulate nociceptive transmission in the spinal cord and contribute to persistent pain, but their role in chronic postoperative pain has not been studied. Here, we ablated pronociceptive neurokinin-1 receptor (NK-1R)-expressing neurons in the rat rostral ventromedial medulla (RVM) to identify their role in CPTP. Cells were ablated by microinjection of the neurotoxin Sar, Met(O2)-Substance P (SSP-SAP), either 2 to 3 weeks before ("Prevention" condition) or 10 days after ("Reversal" condition) thoracotomy with rib retraction. Inactive Blank-SAP was the control. Tactile hypersensitivity was defined by lowered force thresholds for nocifensive responses to von Frey filaments applied over the dorsal trunk, and pain-like behavior assessed by the Qualitative Hyperalgesia Profile; both were followed for 5 weeks after surgery. SSP-SAP injection before surgery resulted in ∼95% loss of NK-1R neurons in RVM and prevented postoperative mechano-hypersensitivity. Blank-SAP was ineffective. SSP-SAP given at postoperative day 10 was equally effective in ablating NK-1R neurons but fully reversed mechano-hypersensitivity in only 3 of 9 hypersensitive rats. Fewer rats showed intense pain-like behavior, by Qualitative Hyperalgesia Profile analysis, in the Prevention than in the Control conditions, and the more intense pain behaviors declined along with SSP-SAP-induced Reversal of hypersensitivity. Neurokinin-1 receptor-expressing neurons in RVM appear essential for the development but contribute only partially to the maintenance of CPTP.

Thoracotomy results in a high frequency of chronic postoperative pain. Resolvins are endogenous molecules, synthesized and released by activated immune cells, effective against inflammatory and neuropathic pain. Different resolvins have differential actions on selective neuronal and glial receptors and enzymes. This article examines the ability of intrathecal resolvin D1 and resolvin D2 to reduce chronic post-thoracotomy pain in rats. Thoracotomy, involving intercostal incision and rib retraction, resulted in a decrease in the mechanical force threshold to induce nocifensive behavior, an enlargement of the pain-sensitive area, and an increase in the fraction of rats showing nocifensive behavior, all for at least 5 weeks. The qualitative nature of the behavioral responses to tactile stimulation changed dramatically after thoracotomy, including the appearance of vigorous behaviors, such as turning, shuddering, and squealing, all absent in naive rats. Intrathecal delivery of resolvin D1 (30 ng/30 μL), at surgery or 4 days later, halved the spread of the mechanosensitive area, lowered by 60% the percent of rats with tactile hypersensitivity, and reduced the drop in threshold for a nocifensive response, along with a reduction in the occurrence of vigorous nocifensive responses. Resolvin D2's actions on threshold changes were statistically the same. These findings suggest that intrathecal resolvins, delivered preoperatively or several days later, can prevent chronic postoperative hyperalgesia., Perspective: In studies of rats, the injection of the proresolving compounds of the resolvin-D series into spinal fluid, before or just after thoracotomy surgery, prevents the occurrence of acute and chronic pain. If these chemicals, which have shown no side-effects, were used in humans it might greatly reduce chronic postoperative pain., (Copyright © 2017 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Background: Antidepressant S-duloxetine alleviates intractable pain associated with diabetic peripheral neuropathy and fibromyalgia. It also reduces both acute and persistent pain in various animal models. This study addresses whether the enantiomer, R-duloxetine, and the homolog, N-methyl duloxetine, could act as analgesics and whether they block neuronal Na⁺ channels., Methods: The rat incision plus extension model on the dorsothoracic skin was applied to evoke postoperative mechanoallodynia and hyperalgesia, measured for 5 days postoperatively by local responses to von Frey filaments. R-Duloxetine and N-methyl duloxetine were administered systemically (intraperitoneal) or locally (subcutaneous [SC]) 1 hour before the surgery. The block of Na currents in rat neuronal GH3 cells was determined under the whole-cell configuration., Results: Ipsilateral SC injections (2 mg/0.4 mL) of R-duloxetine and N-methyl duloxetine reduced both postoperative allodynia and hyperalgesia by approximately 89% to 99% in the area under the curve of skin responses next to incision over 5 days. Systemic intraperitoneal injections at a higher dosage (10 mg) had smaller analgesic effects (reduced by approximately 53%-69%), whereas contralateral SC injections (10 mg) were ineffective. Both R-duloxetine and N-methyl duloxetine blocked neuronal Na⁺ currents, with a higher affinity for the inactivated than the resting states. In addition, both drugs elicited significant use-dependent block of Na currents when stimulated at 5 Hz., Conclusions: R-Duloxetine and N-methyl duloxetine are highly effective against postoperative pain using the skin incision model, and they elicit both tonic and use-dependent block of neuronal Na⁺ channels. Our results suggest that R-duloxetine and N-methyl duloxetine are applicable as novel analgesics.

Background: Contralateral hyperalgesia, occurring after unilateral injury, is usually explained by central sensitization in spinal cord and brain. We previously reported that injection of endothelin-1 (ET-1) into one rat hindpaw induces prolonged mechanical and chemical sensitization of the contralateral hindpaw. Here, we examined the role of contralateral efferent activity in this process., Methods: ET-1 (2 nmol, 10 μL) was injected subcutaneously into the plantar surface of right (ipsilateral) hindpaw (ILP), and the thermal response latency and mechanical threshold for nocifensive withdrawal were determined by the use of, respectively, plantar radiant heating and von Frey filaments, for both ILP and contralateral hindpaws (CLP). Either paw was anesthetized for 60 minutes by direct injection of bupivacaine (0.25%, 40 μL), 30 minutes before ET-1. Alternatively, the contralateral sciatic nerve was blocked for 6 to 12 hours by percutaneous injection of bupivacaine-releasing microspheres 30 minutes before injection of ET-1. Systemic actions of these bupivacaine formulations were simulated by subcutaneous injection at the nuchal midline., Results: After the injection of ET-1, the mechanical threshold of both ILP and CLP decreased by 2 hours, appeared to be lowest around 24 hours, and recovered through 48 hours to preinjection baseline at 72 hours. These hypersensitive responses were suppressed by bupivacaine injected into the ipsilateral paw before ET-1. Injection of the CLP by bupivacaine also suppressed the hypersensitivity of the CLP at all test times, and that of the ILP, except at 2 hours when it increased the sensitivity. This same pattern of change occurred when the contralateral sciatic nerve was blocked by bupivacaine-releasing microspheres. The systemic actions of these bupivacaine formulations were much smaller and only reached significance at 24 hours post-ET-1. Thermal hypersensitivity after ET-1 injection also occurred in both ILP and CLP and showed the same pattern in response to the 2 contralateral anesthetic procedures., Conclusions: These results show that efferent transmission through the contralateral innervation into the paw is necessary for contralateral sensitization by ET-1, suggesting that the release of substances by distal nerve endings is involved. The release of substances in the periphery is essential for contralateral sensitization by ET-1 and may also contribute to secondary hyperalgesia, occurring at loci distant from the primary injury, that occurs after surgery or nerve damage.

Chronic pain after surgery limits social activity, interferes with work, and causes emotional suffering. A major component of such pain is reported as resting or spontaneous pain with no apparent external stimulus. Although experimental animal models can simulate the stimulus-evoked chronic pain that occurs after surgery, there have been no studies of spontaneous chronic pain in such models. Here the conditioned place preference (CPP) paradigm was used to reveal resting pain after experimental thoracotomy. Male Sprague Dawley rats received a thoracotomy with 1-hour rib retraction, resulting in evoked tactile hypersensitivity, previously shown to last for at least 9 weeks. Intraperitoneal injections of morphine (2.5 mg/kg) or gabapentin (40 mg/kg) gave equivalent 2- to 3-hour-long relief of tactile hypersensitivity when tested 12 to 14 days postoperatively. In separate experiments, single trial CPP was conducted 1 week before thoracotomy and then 12 days (gabapentin) or 14 days (morphine) after surgery, followed the next day by 1 conditioning session with morphine or gabapentin, both versus saline. The gabapentin-conditioned but not the morphine-conditioned rats showed a significant preference for the analgesia-paired chamber, despite the equivalent effect of the 2 agents in relieving tactile allodynia. These results show that experimental thoracotomy in rats causes spontaneous pain and that some analgesics, such as morphine, that reduce evoked pain do not also relieve resting pain, suggesting that pathophysiological mechanisms differ between these 2 aspects of long-term postoperative pain. Perspective: Spontaneous pain, a hallmark of chronic postoperative pain, is demonstrated here in a rat model of experimental postthoracotomy pain, further validating the use of this model for the development of analgesics to treat such symptoms. Although stimulus-evoked pain was sensitive to systemic morphine, spontaneous pain was not, suggesting different mechanistic underpinnings., (Copyright © 2015 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Background: Endothelin-1 (ET-1) induces pain-like behavior in animals and man by activating the Gq protein-coupled receptor endothelin-A (ETA ). Activation of ETA receptors on nociceptor membranes evokes intracellular calcium transients and alters membrane Na(+) and K(+) channel and TRPV1 currents, leading to neuronal hyper-excitability manifested by spontaneous and evoked pain behaviors in vivo. In addition to blocking sodium channels, local anesthetics inhibit the Gq protein-coupled signaling of several inflammatory and pro-algesic mediators. In this study, we aimed to investigate the actions of local anesthetics on ETA -mediated increases in intracellular calcium in ND7/104 model sensory neurons., Methods: Increases in intracellular calcium were measured by the fluorescent indicator fura-2 in a sensory neuron-derived cell line (ND7/104), which endogenously expresses ETA receptors. Effects of lidocaine and bupivacaine, along with their respective membrane-impermeant derivatives QX-314, LEA-123 and LEA-124, on peak calcium responses to ET-1 were measured., Results: Bupivacaine suppressed ET-1 responses in a concentration-dependent and non-competitive manner with an IC50 of 3.79 ± 1.63 mM. Bupivacaine (6 mM) reduced the Emax for ET-1 from 50.07 ± 1.91 mM to 27.30 ± 2.92 mM. The actions of bupivacaine occurred quickly and were rapidly reversible. Membrane-impermeant analogs of bupivacaine (LEA-123 and LEA-124, 6 mM) were without effect, as was lidocaine (10 mM) and its quaternary derivative QX-314 (10 mM)., Conclusion: Bupivacaine inhibits ETA -mediated calcium transients at clinically relevant concentrations through an intracellular target. The anti-inflammatory and analgesic actions of bupivacaine may be at least partially due to its inhibitory action on Gq -coupled receptors, including ETA., (© 2015 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Background: Duloxetine is an antidepressant effective for major depressive disorder and also the alleviation of pain for patients with diabetic peripheral neuropathy, chronic musculoskeletal pain, and fibromyalgia. How duloxetine works in pain relief remains unknown. In this study, we address whether duloxetine could act as an analgesic via systemic and local applications., Methods: Efficacies of bupivacaine and duloxetine applied subcutaneously at the incision site against acute postoperative pain were compared after rat skin incision. Contralateral and intraperitoneal injections were used to assess systemic efficacy of duloxetine. Local anesthetic actions were assayed through functional block of the rat sciatic nerve. Inhibition by duloxetine of neuronal Na channels was characterized in rat GH3 cells., Results: Our studies showed that subcutaneous duloxetine (2 mg) reduced hyperalgesia and allodynia for several days after skin incision, whereas subcutaneous bupivacaine (2 mg) did not. Contralaterally injected duloxetine (10 mg) had minimal effects on postoperative pain. Intraperitoneal duloxetine also reduced both allodynia and hyperalgesia, albeit at higher doses (10-20 mg). Duloxetine (2 mg) inhibited motor and nociceptive functions via sciatic nerve block for approximately 24 hours. It also reduced Na currents with 50% inhibitory concentrations of 30.4 ± 1.2 μM and 4.26 ± 0.19 μM (n = 8) for resting and fast-inactivated channels, respectively. Furthermore, duloxetine (10 μM) elicited additional use-dependent block of peak Na currents by approximately 70% when stimulated at 5 Hz., Conclusions: Our results demonstrate that duloxetine can act as a local anesthetic and an analgesic drug via both local and systemic applications. Because duloxetine inhibits neuronal Na currents with high potency, it may exert its antihyperalgesic effects through inhibition of the spontaneous nerve impulses that result from peripheral injury, encompassing its actions on multiple central nervous system and peripheral targets.

Background: Postoperative pain is treated incompletely and ineffectively in many circumstances, and chronic postoperative pain causes suffering and diminishes productivity. The objective of this project is to determine whether a recently developed slow-release formulation of bupivacaine was effective in reducing the experimental chronic postoperative pain., Methods: In male Sprague-Dawley rats (250-300 g body weight), bupivacaine-releasing microspheres (MS-Bupi), containing 60 mg of bupivacaine base, were locally injected (MS-Bupi-L) 2 hours preoperatively into the subcutaneous compartment at the locus for experimental thoracotomy. Hypersensitivity to tactile stimulation was assessed by reductions in the threshold force required to induce a response to von Frey filaments (VFH) applied to the hairy back near the incision/retraction site. Pain behavior was assessed using a Qualitative Hyperalgesia Profile. Control groups included rats receiving the same dose of MS-Bupi but at a distant site on the back (MS-Bupi-D, testing for systemic drug actions) and rats receiving the same mass of microspheres with no drug (MS-Placebo) at the wound site. Rats were tested for 3 days before and 28 days (postoperative days [PODs]) after the procedure. Withdrawal threshold differences, which were the primary outcome measure, among all treatment groups were assessed by the Kruskal-Wallis test, after which pairwise comparisons were made by determining Wilcoxon-Mann-Whitney odds (WMWodds), with Bonferroni correction of the confidence intervals., Results: Microsphere bupivacaine released near the incision reduced the chronic tactile allodynia after thoracotomy. The threshold values during PODs 14 to 28 were different among the 3 treatment groups when examined on PODs 14, 16, 18, 23, 25, and 28 but not on POD21 (P = 0.0603). WMWodds showed that threshold of the MS-Bupi-L group differed from those of the MS-Bupi-D and the MS-Placebo groups for all the tested PODs, whereas the thresholds of the MS-Bupi-D group never differed from those of the MS-Placebo group. Area-under-curve analysis for threshold reductions below baseline, using WMWodds, also showed a reduction during the entire 28 PODs that was greater for the MS-Bupi-L group compared with the MS-Placebo or MS-Bupi-D group. The incidence of intense pain scores by the Qualitative Hyperalgesia Profile analysis was observed in 7 of 8 rats in the MS-Placebo group and in 5 of 8 rats in the MS-Bupi-L group., Conclusions: Local slow release of bupivacaine subcutaneously from the MS-Bupi formulation suppresses postoperative mechanical hypersensitivity for ≥4 weeks after experimental thoracotomy. Systemic bupivacaine from this treatment has no effect on this hypersensitivity.

Objective: To review the published literature regarding the effects of anesthesia on cancer surgery to prevent tumor cell proliferation/migration or induce apoptosis., Background: Surgery is the main treatment for potentially curable solid tumors, but most cancer-related deaths in patients who have received previous surgical treatment are caused by metastatic disease. There is increasing evidence that anesthetic technique has the potential to affect long-term outcome after cancer surgery., Methods: This work reviews the English published literature that was obtained by performing a search of the PubMed database up to January 2014. We selected articles that provided evidence or reviewed the possible actions of anesthetics on cancer cells or the influence of anesthesia in recurrence/outcome., Results: Inhaled anesthetics induce immunosuppression and activate inflammatory cascade activation, whereas propofol has a protective action. Opioids might promote cancer recurrence and metastasis. In vitro and in vivo studies have demonstrated that local anesthetics inhibit proliferation and migration of cancer cells and induce apoptosis., Conclusions: Anesthesiologists should follow current best clinical practice and include all strategies that effectively decrease pain and attenuate stress. Regional anesthesia and multimodal analgesia, adding anti-inflammatory drugs, play an unquestionable role in the control of perioperative pain and may improve recurrence-free survival., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Antibodies are important tools for the study of protein expression but are often used without full validation. In this study, we used Western blots to characterize antibodies targeted to the N or C terminal (NT or CT, respectively) and the second or third intracellular loop (IL2 or IL3, respectively) of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50-kDa band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37-kDa band but failed to detect endogenous ETB in rat brain. Bands detected by the CT- or IL3-targeted antibody were found to be unrelated to ETB. Our findings show that functional ETB can be detected at 50 or 37kDa on Western blot, with drastic differences in antibody affinity for these bands. The 37-kDa band likely reflects ETB processing, which appears to be dependent on cell type and/or culture condition., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Endothelin (ET-1), an endogenous peptide with a prominent role in cutaneous pain, causes mechanical hypersensitivity in the rat hind paw, partly through mechanisms involving local release of algogenic molecules in the skin. The present study investigated involvement of cutaneous ATP, which contributes to pain in numerous animal models. Pre-exposure of ND7/104 immortalized sensory neurons to ET-1 (30nM) for 10min increased the proportion of cells responding to ATP (2μM) with an increase in intracellular calcium, an effect prevented by the ETA receptor-selective antagonist BQ-123. ET-1 (3nM) pre-exposure also increased the proportion of isolated mouse dorsal root ganglion neurons responding to ATP (0.2-0.4μM). Blocking ET-1-evoked increases in intracellular calcium with the IP3 receptor antagonist 2-APB did not inhibit sensitization to ATP, indicating a mechanism independent of ET-1-mediated intracellular calcium increases. ET-1-sensitized ATP calcium responses were largely abolished in the absence of extracellular calcium, implicating ionotropic P2X receptors. Experiments using quantitative polymerase chain reaction and receptor-selective ligands in ND7/104 showed that ET-1-induced sensitization most likely involves the P2X4 receptor subtype. ET-1-sensitized calcium responses to ATP were strongly inhibited by broad-spectrum (TNP-ATP) and P2X4-selective (5-BDBD) antagonists, but not antagonists for other P2X subtypes. TNP-ATP and 5-BDBD also significantly inhibited ET-1-induced mechanical sensitization in the rat hind paw, supporting a role for purinergic receptor sensitization in vivo. These data provide evidence that mechanical hypersensitivity caused by cutaneous ET-1 involves an increase in the neuronal sensitivity to ATP in the skin, possibly due to sensitization of P2X4 receptors., (Copyright © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Background and Objectives: Postoperative pain alters physiological functions and delays discharge. Perioperative local anesthetics are effective analgesics in the immediate 1- to 2-day postoperative period, but acute pain often lasts longer. The goal of this work was to develop a local anesthetic formulation adhering to an intraoperative implanted device that reduces pain for at least 3 days after surgery., Methods: Six groups, each with 8 rats, were studied. In a control group (group I), one 1.2-cm-long incision of the skin was followed by blunt dissection to separate the skin away from the underlying tissues and closing with 2 sutures. In 3 of the treatment groups, the same surgical procedure was used, with the subcutaneous space formed by the blunt dissection lined with a 1-cm square patch of hernia mesh coated with poly lactide co-glycolic acid microspheres containing approximately 17 mg of bupivacaine (group II), no drug (placebo; group III), or bupivacaine free-base powder (group IV). Uncoated mesh implants (group V) served as a secondary control. A standard bupivacaine solution (0.4 mL, 0.5%; 2-mg dose) was infiltrated subcutaneously 30 minutes before the surgery and served as a standard control (group VI). Mechanosensitivity of the skin was tested by the local subcutaneous muscle responses to cutaneous tactile stimulation by von Frey hairs with forces of 4 g (for allodynia) and 15 g (for hyperalgesia) preoperatively and for 7 postoperative days., Results: Control rats (group I) showed mechanohypersensitivity, indicative of postoperative allodynia and hyperalgesia, for all 7 postoperative days. Mechanohyperalgesia in rats that received mesh coated with bupivacaine-releasing microspheres (group II) was reduced during this period to 13% of control postoperative values (P < 0.001); mesh coated with bupivacaine base (group IV) reduced it by 50% (P = 0.034). The placebo mesh (group III) and uncoated mesh (group V) caused no significant reduction of mechanohypersensitivity, and bupivacaine solution infiltrated before the incision (group VI) reduced hypersensitivity for only approximately 2 hours, an overall insignificant effect., Conclusions: Bupivacaine slowly released for 72 hours from microspheres adsorbed to the hernia mesh significantly suppresses evoked postoperative hypersensitivity for at least 1 week and is more effective than a suspension of these microspheres or preoperative single-shot infiltration of bupivacaine.

Background: Acute postoperative pain causes physiological deficits and slows recovery. Reduction of such pain by local anesthetics that are delivered for several days postoperatively is a desirable clinical objective, which is approached by a new formulation and applied in animal studies reported here., Methods: We subcutaneously injected a new formulation of poly-lactic-co-glycolic acid polymer microspheres, which provides steady drug release for 96+ hours into rats at the dorsal region 2 hours before surgery. A single 1.2-cm-long skin incision was followed by blunt dissection of skin away from the underlying fascia, and closed by 2 sutures, followed by 14 days of testing. Microspheres containing 5, 10, 20, and 40 mg bupivacaine were injected locally 2 hours before surgery; bupivacaine-free microspheres were the vehicle control, and bupivacaine HCl solution (0.5%), the positive control. Mechanical sensitivity was determined by the frequency of local muscle contractions to repeated pokes with nylon monofilaments (von Frey hairs) exerting 4 and 15 g forces, testing, respectively, allodynia and hyperalgesia, and by pinprick., Results: Injection of bupivacaine microspheres (40 mg drug) into intact skin reduced responses to 15 g von Frey hairs for 6 hours and to pinprick for 36 hours. Respective reductions from bupivacaine HCl lasted for 3 and 2 hours. Skin incision and dissection alone caused mechanical allodynia and hyperalgesia for 14 days. Microspheres containing 20 or 40 mg bupivacaine suppressed postoperative hypersensitivity for up to 3 days, reduced integrated allodynia (area under curve of response versus time) over postoperative days 1 to 5 by 51% ± 20% (mean ± SE) and 78% ± 12%, and reduced integrated hyperalgesia by 55% ± 13% and 64% ± 11%, for the respective doses. Five and ten milligrams bupivacaine in microspheres and the 0.5% bupivacaine solution were ineffective in reducing postoperative hypersensitivity, as were 40 mg bupivacaine microspheres injected contralateral to the incision., Conclusions: Significant suppression of postoperative pain by the slow-release bupivacaine preparation outlasts its anesthetic action on intact skin. These findings demonstrate preventive analgesia and indicate the importance of acute processes in the development of chronic postoperative pain.

Thoracotomy often results in chronic pain, characterized by resting pain and elevated mechano-sensitivity. This paper defines complex behavioral responses to tactile stimulation in rats after thoracotomy, shown to be reversibly relieved by systemic morphine, in order to develop a novel qualitative "pain" score. A deep incision and 1 hour of rib retraction in male Sprague-Dawley rats resulted in reduced threshold and a change in the locus of greatest tactile (von Frey filament) sensitivity, from the lower back to a more rostral location around the wound site, and extending bilaterally. The fraction of rats showing nocifensive responses to mild stimulation (10 gm) increased after thoracotomy (from a pre-operative value of 0/10 to 8/10 at 10 days post-op), and the average threshold decreased correspondingly, from 15 gm to ∼4 gm. The nature of the nocifensive responses to tactile stimulation, composed pre-operatively only of no response (Grade 0) or brief contractions of the local subcutaneous muscles (Grade I), changed markedly after thoracotomy, with the appearance of new behaviors including a brisk lateral "escape" movement and/or a 180° rotation of the trunk (both included as Grade II), and whole body shuddering, and scratching and squealing (Grade III). Systemic morphine (2.5 mg/kg, i.p.) transiently raised the threshold for response and reduced the frequency of Grade II and III responses, supporting the interpretation that these represent pain. The findings support the development of a Qualitative Hyperalgesic Profile to assess the complex behavior that indicates a central integration of hyperalgesia.

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease.

Knowledge about the mechanism of impulse blockade by local anesthetics has evolved over the past four decades, from the realization that Na + channels were inhibited to affect the impulse blockade to an identification of the amino acid residues within the Na + channel that bind the local anesthetic molecule. Within this period appreciation has grown of the state-dependent nature of channel inhibition, with rapid binding and unbinding at relatively high affinity to the open state, and weaker binding to the closed resting state. Slow binding of high affinity for the inactivated state accounts for the salutary therapeutic as well as the toxic actions of diverse class I anti-arrhythmic agents, but may have little importance for impulse blockade, which requires concentrations high enough to block the resting state. At the molecular level, residues on the S6 transmembrane segments in three of the homologous domains of the channel appear to contribute to the binding of local anesthetics, with some contribution also from parts of the selectivity filter. Binding to the inactivated state, and perhaps the open state, involves some residues that are not identical to those that bind these drugs in the resting state, suggesting spatial flexibility in the "binding site". Questions remaining include the mechanism that links local anesthetic binding with the inhibition of gating charge movements, and the molecular nature of the theoretical "hydrophobic pathway" that may be critical for determining the recovery rates from blockade of closed channels, and thus account for both therapeutic and cardiotoxic actions.

Endothelin-1 (ET-1) is a pain mediator, elevated in skin after injury, which potentiates noxious thermal and mechanical stimuli (hyperalgesia) through the activation of ET(A) (and, perhaps, ET(B)) receptors on pain fibers. Part of the mechanism underlying this effect has recently been shown to involve potentiation of neuronal TRPV1 by PKCɛ. However, the early steps of this pathway, which are recapitulated in HEK 293 cells co-expressing TRPV1 and ET(A) receptors, remain unexplored. To clarify these steps, we investigated the pharmacological profile and signaling properties of native endothelin receptors in immortalized cell lines including HEK 293 and ND7 model sensory neurons. Previously we showed that in ND7/104, a dorsal root ganglia-derived cell line, ET-1 elicits a rise in intracellular calcium ([Ca(2+)](in)) which is blocked by BQ-123, an ET(A) receptor antagonist, but not by BQ-788, an ET(B) receptor antagonist, suggesting that ET(A) receptors mediate this effect. Here we extend these findings to HEK 293T cells. Examination of the expression of ET(A) and ET(B) receptors by RT-PCR and [(125)I]-ET-1 binding experiments confirms the slight predominance of ET(A) receptor binding sites and messenger RNA in both ND7/104 and HEK 293T cells. In addition, selective agonists of the ET(B) receptor (sarafotoxin 6c, BQ-3020 or IRL-1620) do not induce a transient increase in [Ca(2+)](in). Furthermore, reduction of ET(B) mRNA levels by siRNA does not abrogate calcium mobilization by ET-1 in HEK 293T cells, corroborating the lack of an ET(B) receptor role in this response. However, in HEK 293 cells with low endogenous ET(A) mRNA levels, ET-1 does not induce a transient increase in [Ca(2+)](in). Observation of the [Ca(2+)](in) elevation in ND7/104 and HEK 293T cells in the absence of extracellular calcium suggests that ET-1 elicits a release of calcium from intracellular stores, and pretreatment of the cells with pertussis toxin or a selective inhibitor of phospholipase C (PLC) point to a mechanism involving Gαq/11 coupling. These results are consistent with the hypothesis that a certain threshold of ET(A) receptor expression is necessary to drive a transient [Ca(2+)](in) increase in these cells and that this process involves release of calcium from intracellular stores following Gαq/11 activation., (2011 Elsevier Ltd. All rights reserved.)

A profound tachyphylaxis of the acute nocifensive flinching (pain) response to subcutaneous injection of endothelin-1 (ET-1) into the hind paw footpad is shown by the reduced response to a second injection. Flinching from the second injection was 20% +/- 5%, 57% +/- 18%, 79% +/- 35%, and 100% +/- 17% of that from the first injection (both 200 micromol/L, 2 nmol) at respective intervals of 24, 30, 48, and 72 h. Inhibition of afferent impulses by local anesthesia of the sciatic nerve, reducing initial flinching to 6%-13% of control, did not affect the tachyphylaxis for the second injection at 24 h. There was no cross-desensitization between formalin and ET-1 injected sequentially into the same paw. Suppression of descending inhibitory effects from endogenous opiates by naloxone (5-8 mg/kg, i.p.), given 30 min before the second ET-1 injection, did not prevent tachyphylaxis. Diffuse effects caused by an initial subcutaneous ET-1 injection into the tail or forepaw resulted in sensitization of the response to ET-1 in the hind paw, rather than tachyphylaxis. In contrast, selective inhibition of local ETA receptors during the initial administration of ET-1, by the antagonist BQ-123 (3.2 mmol/L), reduced tachyphylaxis of nocifensive flinching. Therefore, prolonged pain tachyphylaxis is not due to reduced responsiveness of the CNS, but rather depends on the functional sensitivity or availability of peripheral ET(A) receptors.

Subcutaneous injection of the peptide endothelin-1 (ET-1) into the rat's footpad is known to cause rapid, transient ipsilateral mechanical and thermal sensitization and nocifensive hind paw flinching. Here we report that local injection of ET-1 (2 nmoles) into one hind paw slowly sensitizes the contralateral paw to chemical and mechanical stimulation. There was a 1.5-2-fold increase in the hind paw flinching response, over that from the first injection, to a second injection of the same dose of ET-1 delivered 24 h later into the contralateral paw. A similar increase in the number of flinches during the second phase of the response to formalin also occurred in the contralateral paw 24 h after ET-1. The contralateral paw withdrawal threshold to von Frey hairs was lowered by approximately 55% at 24 h after ipsilateral ET-1 injection. ET-1 injected s.c. at a segmentally unrelated location, the nuchal midline, caused no sensitization of the paws, obviating a systemic route of action. Local anesthetic block of the ipsilateral sciatic nerve during the period of initial response to ipsilateral ET-1 prevented contralateral sensitization, indicating the importance of local afferent transmission, although ipsilateral desensitization was not changed. These findings suggest that peripheral ET-1 actions lead to central sensitization that alters responses to selected stimuli.

Unlabelled: Subcutaneous injection of endothelin-1 (ET-1) into the glabrous skin of the rat's hind paw is known to produce impulses in nociceptors and acute nocifensive behavioral responses, such as hind paw flinching, and to sensitize the skin to mechanical and thermal stimulation. In this report, we show that in contrast to the responses in glabrous skin, ET-1 injected subcutaneously into rat hairy skin causes transient antinociception. Concentrations of 1 to 50 microM ET-1 (in 0.05 mL) depress the local nocifensive response to noxious tactile probing at the injection site with von Frey filaments for 30 to 180 minutes; distant injections have no effect at this site, showing that the response is local. Selective inhibition of ET(A) but not of ET(B) receptors inhibits this antinociception, as does coinjection with nimodipine (40 muM), a blocker of L-type Ca(2+) channels. Local subcutaneous injection of epinephrine (45 microM) also causes antinociception through alpha-1 adrenoreceptors, but such receptors are not involved in the ET-1-induced effect. Both epinephrine and ET-1, at antinociceptive concentrations, reduce blood flow in the skin; the effect from ET-1 is largely prevented by subcutaneous nimodipine. These data suggest that ET-1-induced antinociception in the hairy skin of the rat involves cutaneous vasoconstriction, presumably through neural ischemia, resulting in conduction block., Perspective: The pain-inducing effects of ET-1 have been well documented in glabrous skin of the rat, a frequently used test site. The opposite behavioral effect, antinociception, occurs from ET-1 in hairy skin and is correlated with a reduction in blood flow. Vasoactive effects are important in assessing mechanisms of peripherally acting agents.

Mitogen-activated protein kinases (MAPKs) are important for intracellular signal transduction and play critical roles in regulating neural plasticity and inflammatory responses. The MAPK family consists of three major members: extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK), which represent three separate signaling pathways. Accumulating evidence shows that all three MAPK pathways contribute to pain sensitization after tissue and nerve injury via distinct molecular and cellular mechanisms. Activation (phosphorylation) of MAPKs under different persistent pain conditions results in the induction and maintenance of pain hypersensitivity via non-transcriptional and transcriptional regulation. In particular, ERK activation in spinal cord dorsal horn neurons by nociceptive activity, via multiple neurotransmitter receptors, and using different second messenger pathways plays a critical role in central sensitization by regulating the activity of glutamate receptors and potassium channels and inducing gene transcription. ERK activation in amygdala neurons is also required for inflammatory pain sensitization. After nerve injury, ERK, p38, and JNK are differentially activated in spinal glial cells (microglia vs astrocytes), leading to the synthesis of proinflammatory/pronociceptive mediators, thereby enhancing and prolonging pain. Inhibition of all three MAPK pathways has been shown to attenuate inflammatory and neuropathic pain in different animal models. Development of specific inhibitors for MAPK pathways to target neurons and glial cells may lead to new therapies for pain management. Although it is well documented that MAPK pathways can increase pain sensitivity via peripheral mechanisms, this review will focus on central mechanisms of MAPKs, especially ERK.

The contribution of endothelin-1 (ET-1), acting via endothelin-A receptors (ET(A)), on post-incisional pain was examined in a rat model of incision through the hairy skin of the lumbar dorsum. Post-incisional mechanical hyperesthesia was evaluated by cutaneous trunci muscle reflexes (CTMR) of subcutaneous muscles responding to stimulation with von Frey filaments near the wound (primary responses) and at a distance, especially on the contralateral dorsum (secondary responses, involving spinal circuits). The role of ET(A) was determined by pre-incisional, subcutaneous injection of the selective receptor antagonist BQ-123 at the incision site, 15 min or 24h before surgery. Control incisions showed both primary tactile allodynia and hyperalgesia, and a weaker secondary hyperesthesia, peaking 3-4h after surgery and lasting at least 24h. Primary allodynia, but not hyperalgesia, was dose-dependently suppressed by 15 min pre-incisional BQ-123. In contrast, both secondary allodynia and hyperalgesia were inhibited by local BQ-123. The suppression of primary allodynia by local antagonist disappeared in 24h, but that of secondary hyperesthesia remained strong for at least 24h. Systemically delivered BQ-123 was without effect on any post-incisional hyperesthesia, and if the antagonist was locally injected 24h before surgery there was no difference on hyperesthesia compared to vehicle injected at that time. We conclude that ET-1, released from skin by incision, activates nociceptors to cause primary allodynia and to sensitize spinal circuits through central sensitization. Blockade of ET(A) in the immediate peri-operative period prevents the later development of central sensitization.

Background: Sandhoff disease is an inherited lysosomal storage disease caused by a mutation in the gene for the beta-subunit (Hexb gene) of beta-hexosaminidase A (alphabeta) and B (beta beta). The beta-subunit together with the GM2 activator protein catabolize ganglioside GM2. This enzyme deficiency results in GM2 accumulation primarily in the central nervous system. To investigate how abnormal GM2 catabolism affects the peripheral nervous system in a mouse model of Sandhoff disease (Hexb-/-), we examined the electrophysiology of dissected sciatic nerves, structure of central and peripheral myelin, and lipid composition of the peripheral nervous system., Results: We detected no significant difference in signal impulse conduction velocity or any consistent change in the frequency-dependent conduction slowing and failure between freshly dissected sciatic nerves from the Hexb+/- and Hexb-/- mice. The low-angle x-ray diffraction patterns from freshly dissected sciatic and optic nerves of Hexb+/- and Hexb-/- mice showed normal myelin periods; however, Hexb-/- mice displayed a approximately 10% decrease in the relative amount of compact optic nerve myelin, which is consistent with the previously established reduction in myelin-enriched lipids (cerebrosides and sulfatides) in brains of Hexb-/- mice. Finally, analysis of lipid composition revealed that GM2 content was present in the sciatic nerve of the Hexb-/- mice (undetectable in Hexb+/-)., Conclusion: Our findings demonstrate the absence of significant functional, structural, or compositional abnormalities in the peripheral nervous system of the murine model for Sandhoff disease, but do show the potential value of integrating multiple techniques to evaluate myelin structure and function in nervous system disorders.

Interactions of the local anesthetic tetracaine with unilamellar vesicles made of dimyristoyl or dipalmitoyl phosphatidylcholine (DMPC or DPPC), the latter without or with cholesterol, were examined by following changes in the drug's fluorescent properties. Tetracaine's location within the membrane (as indicated by the equivalent dielectric constant around the aromatic fluorophore), its membrane:buffer partition coefficients for protonated and base forms, and its apparent pK(a) when adsorbed to the membrane were determined by measuring, respectively, the saturating blue shifts of fluorescence emission at high lipid:tetracaine, the corresponding increases in fluorescence intensity at this lower wavelength with increasing lipid, and the dependence of fluorescence intensity of membrane-bound tetracaine (TTC) on solution pH. Results show that partition coefficients were greater for liquid-crystalline than solid-gel phase membranes, whether the phase was set by temperature or lipid composition, and were decreased by cholesterol; neutral TTC partitioned into membranes more strongly than the protonated species (TTCH(+)). Tetracaine's location in the membrane placed the drug's tertiary amine near the phosphate of the headgroup, its ester bond in the region of the lipids' ester bonds, and associated dipole field and the aromatic moiety near fatty acyl carbons 2-5; importantly, this location was unaffected by cholesterol and was the same for neutral and protonated tetracaine, showing that the dipole-dipole and hydrophobic interactions are the critical determinants of tetracaine's location. Tetracaine's effective pK(a) was reduced by 0.3-0.4 pH units from the solution pK(a) upon adsorption to these neutral bilayers, regardless of physical state or composition. We propose that the partitioning of tetracaine into solid-gel membranes is determined primarily by its steric accommodation between lipids, whereas in the liquid-crystalline membrane, in which the distance between lipid molecules is larger and steric hindrance is less important, hydrophobic and ionic interactions between tetracaine and lipid molecules predominate.

Local anesthetics are used broadly to prevent or reverse acute pain and treat symptoms of chronic pain. This chapter, on the analgesic aspects of local anesthetics, reviews their broad actions that affect many different molecular targets and disrupt their functions in pain processing. Application of local anesthetics to peripheral nerve primarily results in the blockade of propagating action potentials, through their inhibition of voltage-gated sodium channels. Such inhibition results from drug binding at a site in the channel's inner pore, accessible from the cytoplasmic opening. Binding of drug molecules to these channels depends on their conformation, with the drugs generally having a higher affinity for the open and inactivated channel states that are induced by membrane depolarization. As a result, the effective potency of these drugs for blocking impulses increases during high-frequency repetitive firing and also under slow depolarization, such as occurs at a region of nerve injury, which is often the locus for generation of abnormal, pain-related ectopic impulses. At distal and central terminals the inhibition of voltage-gated calcium channels by local anesthetics will suppress neurogenic inflammation and the release of neurotransmitters. Actions on receptors that contribute to nociceptive transduction, such as TRPV1 and the bradykinin B2 receptor, provide an independent mode of analgesia. In the spinal cord, where local anesthetics are present during epidural or intrathecal anesthesia, inhibition of inotropic receptors, such as those for glutamate, by local anesthetics further interferes with neuronal transmission. Activation of spinal cord mitogen-activated protein (MAP) kinases, which are essential for the hyperalgesia following injury or incision and occur in both neurons and glia, is inhibited by spinal local anesthetics. Many G protein-coupled receptors are susceptible to local anesthetics, with particular sensitivity of those coupled via the Gq alpha-subunit. Local anesthetics are also infused intravenously to yield plasma concentrations far below those that block normal action potentials, yet that are frequently effective at reversing neuropathic pain. Thus, local anesthetics modify a variety of neuronal membrane channels and receptors, leading to what is probably a synergistic mixture of analgesic mechanisms to achieve effective clinical analgesia.

Subcutaneous endothelin-1 (ET-1; 200 microM, 2 nmoles/paw) injected into the rat hind paw, has been shown to cause robust hind paw flinching (HPF) and paw licking, and to induce impulses selectively in primary nociceptors. Here we report that a much lower [ET-1] sensitizes the paw to a nocifensive withdrawal response to tactile stimulation (by von Frey hairs, VFH), a sensitization that involves local TRPV1 receptors. Injection of 10 microM ET-1 (0.1 nmole/paw) causes only marginal HPF but rapidly (20 mins after injection) lowers the force threshold for paw withdrawal (PWT) to VFH, to approximately 30% of pre-injection baseline. Such tactile allodynia persists for 3 hrs. In rats pre-injected with the TRPV1-antagonists capsazepine (CPZ; 1.33 mM) or 5'-iodoresiniferatoxin (I-RTX; 0.13 microM), 15 min before ET-1, a fast initial drop in PWT, as with ET-1 alone, occurs (to 40% or to 19% of baseline, respectively), but this earliest reduction then regresses back to the pre-injection PWT value more rapidly than with ET-1 alone. The recovery of allodynia from the maximum value is about two times faster for ET-1+CPZ and about 4 times faster for ET-1+ I-RTX, compared with that from ET-1 +vehicle (t(1/2) = 130, 60, and 250 mins, respectively). In contrast, spontaneous pain indicated by overt HPF from ET-1 is not attenuated by TRPV1 antagonists. Tactile allodynia is similarly abbreviated by antagonists of both ET(A) (BQ-123, 32 nmoles/paw) and ET(B) (BQ-788, 30 nmoles/paw) receptors, whereas HPF is abolished by this ET(A) antagonist but enhanced by the ET(B) antagonist. We conclude that low ET-1 causes tactile allodynia, which is characterized by a different time-course and pharmacology than ET-1-induced nociception, and that local TRPV1 receptors are involved in the maintenance of this ET-1-induced allodynia but not in the overt algesic action of ET-1.

Unlabelled: Clinical and experimental data indicate that changes in the expression of voltage-gated sodium channels play a key role in the pathogenesis of neuropathic pain and that drugs that block these channels are potentially therapeutic. Clinical and experimental data also suggest that changes in voltage-gated sodium channels may play a role in inflammatory pain, and here too sodium-channel blockers may have therapeutic potential. The sodium-channel blockers of interest include local anesthetics, used at doses far below those that block nerve impulse propagation, and tricyclic antidepressants, whose analgesic effects may at least partly be due to blockade of sodium channels. Recent data show that local anesthetics may have pain-relieving actions via targets other than sodium channels, including neuronal G protein-coupled receptors and binding sites on immune cells. Some of these actions occur with nanomolar drug concentrations, and some are detected only with relatively long-term drug exposure. There are 9 isoforms of the voltage-gated sodium channel alpha-subunit, and several of the isoforms that are implicated in neuropathic and inflammatory pain states are expressed by somatosensory primary afferent neurons but not by skeletal or cardiovascular muscle. This restricted expression raises the possibility that isoform-specific drugs might be analgesic and lacking the cardiotoxicity and neurotoxicity that limit the use of current sodium-channel blockers., Perspective: Changes in the expression of neuronal voltage-gated sodium channels may play a key role in the pathogenesis of both chronic neuropathic and chronic inflammatory pain conditions. Drugs that block these channels may have therapeutic efficacy with doses that are far below those that impair nerve impulse propagation or cardiovascular function.

Optimal management of neuropathic pain is a major clinical challenge. We investigated the involvement of c-Jun N-terminal kinase (JNK) in neuropathic pain produced by spinal nerve ligation (SNL) (L5). SNL induced a slow (>3 d) and persistent (>21 d) activation of JNK, in particular JNK1, in GFAP-expressing astrocytes in the spinal cord. In contrast, p38 mitogen-activated protein kinase activation was found in spinal microglia after SNL, which had fallen to near basal level by 21 d. Intrathecal infusion of a JNK peptide inhibitor, D-JNKI-1, did not affect normal pain responses but potently prevented and reversed SNL-induced mechanical allodynia, a major symptom of neuropathic pain. Intrathecal D-JNKI-1 also suppressed SNL-induced phosphorylation of the JNK substrate, c-Jun, in spinal astrocytes. However, SNL-induced upregulation of GFAP was not attenuated by spinal D-JNKI-1 infusion. Furthermore, SNL induced a rapid (<12 h) but transient activation of JNK in the L5 (injured) but not L4 (intact) DRG. JNK activation in the DRG was mainly found in small-sized C-fiber neurons. Infusion of D-JNKI-1 into the L5 DRG prevented but did not reverse SNL-induced mechanical allodynia. Finally, intrathecal administration of an astroglial toxin, l-alpha-aminoadipate, reversed mechanical allodynia. Our data suggest that JNK activation in the DRG and spinal cord play distinct roles in regulating the development and maintenance of neuropathic pain, respectively, and that spinal astrocytes contribute importantly to the persistence of mechanical allodynia. Targeting the JNK pathway in spinal astroglia may present a new and efficient way to treat neuropathic pain symptoms.

Endothelin-1 (ET-1) applied to the sciatic nerve or injected into the plantar hindpaw of rats induces pain behavior (ipsilateral hindpaw flinching) and selective excitation of nociceptors by activation of endothelin-A (ET(A)) receptors. To determine the pharmacological profile of the sensory fibers that mediate this pain behavior, we administered lidocaine (LID, a non-selective conduction blocker) or tetrodotoxin (TTX) prior to ET-1. LID (1 or 2%, 0.1 ml) was injected percutaneously into the sciatic notch, or TTX (10 microM, 4 microl) was injected into the sciatic nerve prior to the more distal application of ET-1 (400 microM, 40 microl) onto the sciatic nerve or subcutaneously into the plantar hindpaw (400 microM, 10 microl). LID inhibited ET-1-induced flinching in a dose-dependent manner; the mean total number of flinches was reduced by 39% for 1% LID and by 87% for 2% LID. In contrast, TTX failed to inhibit flinching behavior induced by sciatic nerve application of ET-1 despite a similar magnitude of motor and sensory blockade as that observed with 2% LID. Partial blockade of flinching behavior by intraneural TTX (mean total flinches were reduced by 51%) was observed after subcutaneous injection of ET-1. Unexpectedly, ET-1 prolonged the actions of 1% LID and, even when applied alone, produced clear signs of motor and sensory conduction block. These results are evidence that ET-1-induced pain is transmitted to the central nervous system via sensory fibers using tetrodotoxin-resistant sodium channels, and that ET-1 has analgesic actions that exist despite the activation of local pain pathways.

Background: The adjustment of local anesthetic dosage for peripheral nerve block must meet two basic requirements: a drug concentration sufficient to inhibit Na+ channels to the point of impulse failure and a volume of drug sufficient to expose a length of nerve longer than the "critical length" for propagation failure. This study examines the lidocaine dosage requirement, in milligrams, for functionally assayed sciatic nerve block in the rat using a fourfold range of volume corresponding to concentrations from 2 to 7 mg/ml and compares these blocks with the intraneural lidocaine content after injection of equipotent doses., Methods: Lidocaine was injected percutaneously at the sciatic nerve in volumes of 0.05 ml, 0.1 ml, and 0.2 ml (all at pH 6.8), and quantitative neurobehavioral assays were conducted on male Sprague-Dawley rats weighing from 200 to 250 g. The dose requirements for 50% maximum possible effect (ED50) and for just achieving complete block (i.e., minimal blocking dose for 90% effect), the fraction of completely blocked animals, and the duration of complete block at all doses were assessed for the inhibition of three different functions: proprioception, motor, and nocifensive activities. Radiolabeled (14C) lidocaine was injected in separate experiments, and the total drug content and its longitudinal distribution were determined in nerves dissected from animals (sevoflurane anesthetized) at 10 min, the time of peak block, after injection of the E50 and minimal blocking dose for 100% effect for the three different volumes., Results: For all functions, the smaller the volume, the lower was the E50, resulting in a nearly constant concentration to achieve an equivalent degree of block. Durations of block were longer, and the dose to full block was lower for the smaller injected volumes. Intraneural lidocaine, at the equipotent doses for analgesia, was not related to concentration but rather increased with increasing volume, being almost proportional to the given dose. The longitudinal spread of lidocaine was also greater with increasing volume of lidocaine solution., Conclusion: Blocks of greater depth and longer duration result from injection of smaller volumes and, correspondingly, higher lidocaine concentrations containing the same dose. The corollary is that lower lidocaine doses are required to achieve the same effect when smaller volumes are injected. Curiously, when the equivalent E50 is injected, total drug taken into the nerve is less from the smaller volumes than from the larger volumes, even though the peak functional effects are equal. Total intraneural local anesthetic may not represent the effective drug in the compartment that contains nerve axons, the actual location of neural blockade.

Endothelin-1 (ET-1) is a newly described pain mediator that is involved in the pathogenesis of pain states ranging from trauma to cancer. ET-1 is synthesized by keratinocytes in normal skin and is locally released after cutaneous injury. While it is able to trigger pain through its actions on endothelin-A (ET(A)) receptors of local nociceptors, it can coincidentally produce analgesia through endothelin-B (ET(B)) receptors. Here we map a new endogenous analgesic circuit, in which ET(B) receptor activation induces the release of beta-endorphin from keratinocytes and the activation of G-protein-coupled inwardly rectifying potassium channels (GIRKs, also named Kir-3) linked to opioid receptors on nociceptors. These results indicate the existence of an intrinsic feedback mechanism to control peripheral pain in skin, and establish keratinocytes as an ET(B) receptor-operated opioid pool.

Background and Objectives: Ropivacaine and levobupivacaine, both single S- enantiomers, are being promoted as safer alternatives to racemic bupivacaine. To determine whether levobupivacaine produces a more potent and longer lasting peripheral nerve block than ropivacaine, we compared functional blockade of sciatic nerve in the rat at several doses with these 2 agents., Methods: Percutaneous sciatic nerve blocks were performed in 6 groups of rats (n = 8) with 0.1 mL of 0.0625%, 0.125%, and 0.25% of levobupivacaine or ropivacaine, with and without 1:200,000 epinephrine. In the 2 different hindlegs of the same 8 rats, we measured the degree and duration of deficit of 3 functions of peripheral nerve (proprioception, motor function, nociception) effected by the 2 respective drugs at the same concentration, using a blinded cross-over design., Results: In general, both degree and duration of functional deficits produced by levobupivacaine were slightly greater than those from ropivacaine at the same dose. At the lowest concentration (0.0625%), no functions were completely blocked and the only significant difference was a longer motor impairment by levobupivacaine. At the highest concentration (0.25%), all formulations completely blocked the 3 functions, all of which lasted longer with levobupivacaine than ropivacaine. There were no significant differences in degree and duration of deficits between the 2 drugs at midconcentration (0.125%). The actions of both drugs were generally potentiated by epinephrine, but the rank order of potency was not altered., Conclusions: At the lowest concentration (0.0625%), levobupivacaine produces a greater degree of motor impairment and a longer duration of proprioceptive impairment relative to ropivacaine. At the middle concentration (0.125%), there no differences between the 2 drugs. At the higher concentration (0.25%), which is within the range used clinically for peripheral nerve block (0.25%-0.5%), levobupivacaine produces approximately a 30% longer duration of complete block in each modality compared with that by ropivacaine.

Systemic lidocaine can relieve various forms of neuropathic pain that develop after nerve injury. Mechanical allodynia, defined by a significant drop in paw withdrawal threshold force following spinal nerve ligation (L5-L6) in rats, can be reversed by one 30min lidocaine infusion at a constant plasma concentration as low as 1-2 microg/ml, an effect that is still present when the rats are tested days and weeks afterwards. In this study, we resolved the detailed time course of reversal of ipsilateral and contralateral allodynia in rats with spinal nerve ligation by a single systemic infusion of lidocaine, to 4 microg/ml, given either 2 days after ligation (POD2) or 7 days after ligation (POD7). Male Sprague-Dawley rats were examined for 21 days after undergoing sham operation or spinal nerve ligation to produce allodynia, which was quantified by a lower force of von Frey hairs at the plantar hind paw just required to produce paw withdrawal (paw withdrawal threshold, PWT). Six experimental protocols were followed: rats were infused with lidocaine on POD2 (L2) or on POD7 (L7), or with saline on POD2 (S2) or on POD7 (S7), and sham operated rats were infused with lidocaine on POD2 or on POD7. PWTs were measured during the last 5min of a single 30min lidocaine infusion; at 30, 60, 90, 120, 240 and 360min, and 24, 48 and 72h after beginning infusion, and then every 1-3 days up to 21 days. Three distinct sequential phases of ipsilateral relief were apparent in both L2 and L7 groups: (1) an acute elevation of PWT during the infusion, returning to the pre-infusion allodynic level within 30-60min after infusion; (2) a second, transient elevation of PWT within the next 360min; (3) a sustained elevation of PWT developing slowly over 24h after infusion and maintained over the next 21 days. A significant, although weaker contralateral allodynia developed more slowly (>POD8) than the ipsilateral condition, and could be delayed for more than 2 weeks by lidocaine infusion on POD2 but for only 1 week by the same treatment on POD7. None of the sham operated animals had any allodynic signs and no saline infusions elevated PWT in ligated, allodynic rats. These results of separate phases imply that there are mechanistic differences between the acute relief and the sustained relief of allodynia after a single infusion of lidocaine, and may present an experimental paradigm for investigating the advantages of earlier rather than late therapeutic intervention.

Background: Adding epinephrine to lidocaine solutions for peripheral nerve block potentiates and prolongs the action, but by incompletely understood mechanisms. In an effort to discriminate the pharmacokinetic from the pharmacodynamic effects of epinephrine, the authors measured the lidocaine content of peripheral nerve over the course of block produced by 0.5% lidocaine, with and without epinephrine, and correlated it with the degree of analgesia., Methods: Percutaneous sciatic nerve blocks were performed in 18 groups of rats (10 in each) with 0.1 ml of either 0.5% lidocaine or 0.5% lidocaine with epinephrine (1:100,000). Over the full course of nerve block, the authors regularly measured analgesia to toe pinch and then rapidly removed nerves to assay intraneural lidocaine content at 2-120 min after injection., Results: The kinetics of lidocaine's clearance from nerve was composed of a fast-decaying transient superimposed on a very slowly decaying component. The effect of epinephrine on the intraneural lidocaine content was to increase the amount of lidocaine in the slow-decaying component by threefold to fourfold, although the total neural content was not altered by epinephrine for the first 10 min after injection. Epinephrine prolonged blockade by almost fourfold and enhanced the intensity of peak analgesia, as well as the fraction of rats with complete block, almost throughout the 2-120-min period of behavioral observation., Conclusions: Adding epinephrine to lidocaine solutions increases the intensity and duration of sciatic nerve block in the rat. The early increase in intensity is not matched with an increase in intraneural lidocaine content at these early times, although the prolonged duration of block by epinephrine appears to correspond to an enlarged lidocaine content in nerve at later times, as if a very slowly emptying "effector compartment" received a larger share of the dose. The increase in early analgesia without increased lidocaine content may be explained by a pharmacodynamic action of epinephrine that transiently enhances lidocaine's potency, but also by a pharmacokinetic effect that alters the distribution of the same net content of lidocaine within the nerve.

Understanding the interaction of local anesthetics (LAs) with plasma proteins is essential to understanding their systemic pharmacology and toxicology. The molecular determinants of LA binding to the major variant (F1*S) of human alpha1-acid glycoprotein (AGP) were therefore investigated spectrofluorometrically using whole AGP and a novel, F1*S variant-selective probe previously developed in our laboratory. Equilibrium- competitive displacement of this probe by LAs, observed by the recovery of AGP's fluorescence as the quenching probe was displaced from its high-affinity site, was characterized by inhibitory dissociation constants for the various LAs. The importance of electrostatic factors was assessed by examining the pH dependent binding of an ionizable LA, lidocaine, using the quaternary lidocaine derivative QX-314 [N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium chloride] to control for pH dependent ionization of AGP. Uncharged lidocaine bound with at least 8 times the affinity of protonated lidocaine (K(D) = 4.0 +/- 0.6 microM and >32 microM, respectively). This result is inconsistent with the current model of the AGP-binding site, which depicts a buried pocket having a negatively charged region that interacts with the amino termini of basic drugs. Consistent with the model, however, two sets of structurally homologous LAs (mepivacaine, ropivacaine, bupivacaine, and lidocaine, RAD-240, RAD-241, RAD-242, L-30, W-6603) demonstrated a strong positive correlation between hydrophobicity (measured as the octanol:buffer partition coefficient of the neutral species) and their free energies of dissociation. Given that the tertiary structure of AGP has proven refractory to resolution, these structure-activity studies should contribute to understanding the nature of the binding site on this important protein.

Abnormal impulses caused by very slowly inactivating Na channels of peripheral nerve have been proposed to play a critical role in neuropathic pain. Low concentrations of local anesthetics, often effective in treating experimental and clinical neuropathic pain, are also known to potently suppress the long after-depolarizations induced by these persistently open Na channels. However, these drug actions on impulses that have propagated away from such sites are undetermined. In the present study, the focal application of anemone toxin II (ATX, 300 nM), which slows Na-channel inactivation, produced prolonged depolarizing after-potentials and, coincidentally, induced spontaneous bursting impulse activity that propagated away from the site of ATX application in the frog sciatic nerve in vitro. The application of low concentrations of lidocaine (1-10 microM), both at the site of ATX exposure and at a distant site, selectively and reversibly inhibited the spontaneous bursting, while having no effect on the electrically stimulated initial spike of the compound action potential. Inhibition occurred as a shortening of burst episodes rather than a reduction in frequency of impulses within a burst or a reduction of intraburst impulse amplitude. Tetrodotoxin also inhibited the induced spontaneous activity, but only at concentrations that also depressed the compound action potential spike. These findings show that low concentrations of lidocaine can restore normal firing patterns in nerve where hyperexcitability has been caused by delayed Na-channel inactivation, without acting directly at the site where ectopic impulses are generated. Thus, it appears that the pattern of abnormal activity rather than an abnormally gating Na channel per se can be a target for lidocaine's therapeutic action., (Copyright 2002 International Association for the Study of Pain)

We showed previously that subcutaneous injection of the injury-associated peptide mediator endothelin-1 (ET-1) into the rat plantar hindpaw produces pain behavior and selective excitation of nociceptors, both through activation of ET(A) receptors likely on nociceptive terminals. The potential role of ET(B) receptor activation in these actions of ET-1-has not been examined. Therefore, in these experiments, we studied the effect of blocking or activating ET(B) receptors on ET-1-induced hindpaw flinching and excitation of nociceptors in rats. An ET(B) receptor-selective antagonist, BQ-788 (3 mm), coinjected with ET-1 (200 microm) reduced the time-to-peak of flinching and significantly enhanced the average maximal flinch frequency (MFF). In contrast, coinjection of an ET(B) receptor selective agonist, IRL-1620 (100 or 200 microm), with ET-1 reduced the average MFF and the average total number of flinches. Interestingly, this unexpected inhibitory effect of IRL-1620 was prevented by the nonselective opioid receptor antagonist naloxone (2.75 mm). To confirm these inhibitory actions, we studied the effects of IRL-1620 on ET-1-induced spike responses in single, physiologically characterized nociceptive C-fibers. IRL-1620 suppressed spike responses to ET-1 in all (n = 12) C-units, with mean and maximum response frequencies of 0.08 +/- 0.02 and 1.5 +/- 0.4 impulses/sec versus 0.32 +/- 0.07 and 4.17 +/- 0.17 impulses/sec for ET-1 alone. In additional support of the behavioral results, coinjection of naloxone (2.75 mm) completely prevented this inhibitory action of IRL-1620. These results establish that ET(B) receptor activation inhibits ET-1-induced pain behavior and nociception in a naloxone-sensitive manner and point to a previously unrecognized dual modulation of acute nociceptive signaling by ET(A) and ET(B) receptors in cutaneous tissues.