Association of Interindividual Variation in Plasma Oxytocin With Postcesarean Incisional Pain.

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).

Multiple Inhibitory Mechanisms of Lidocaine on Bradykinin Receptor Activity in Model Sensory Neurons.

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.

A role for neurokinin-1 receptor neurons in the rostral ventromedial medulla in the development of chronic postthoracotomy 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.

Prevention of Chronic Post-Thoracotomy Pain in Rats By Intrathecal Resolvin D1 and D2: Effectiveness of Perioperative and Delayed Drug Delivery.

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.

R-Duloxetine and N-Methyl Duloxetine as Novel Analgesics Against Experimental Postincisional Pain.

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.

Contralateral Hyperalgesia from Injection of Endothelin-1 into the Ipsilateral Paw Requires Efferent Conduction into the Contralateral Paw.

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.

Spontaneous Chronic Pain After Experimental Thoracotomy Revealed by Conditioned Place Preference: Morphine Differentiates Tactile Evoked Pain From Spontaneous Pain.

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.

The Local and Systemic Actions of Duloxetine in Allodynia and Hyperalgesia Using a Rat Skin Incision Pain Model.

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.

Mitigation of experimental, chronic post-thoracotomy pain by preoperative infiltration of local slow-release bupivacaine microspheres.

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.

Cancer surgery: how may anesthesia influence outcome?

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.

Validation of endothelin B receptor antibodies reveals two distinct receptor-related bands on Western blot.

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.

Prolonged amelioration of experimental postoperative pain by bupivacaine released from microsphere-coated hernia mesh.

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.

Sensitization of cutaneous neuronal purinergic receptors contributes to endothelin-1-induced mechanical hypersensitivity.

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.

Inhibition by local bupivacaine-releasing microspheres of acute postoperative pain from hairy skin incision.

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.

Air-stimulated ATP release from keratinocytes occurs through connexin hemichannels.

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.

The Qualitative Hyperalgesia Profile: A New Metric to Assess Chronic Post-Thoracotomy 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.

ET-1 induced Elevation of intracellular calcium in clonal neuronal and embryonic kidney cells involves endogenous endothelin-A receptors linked to phospholipase C through Gα(q/11).

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.

Remarkably long-lasting tachyphylaxis of pain responses to ET-1: evidence against central nervous system involvement.

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.