Neural Selective Cryoneurolysis with Ice Slurry Injection in a Rat Model.

BACKGROUND: Postoperative pain caused by trauma to nerves and tissue around the surgical site is a major problem. Perioperative steps to reduce postoperative pain include local anesthetics and opioids, the latter of which are addictive and have contributed to the opioid epidemic. Cryoneurolysis is a nonopioid and long-lasting treatment for reducing postoperative pain. However, current methods of cryoneurolysis are invasive, technically demanding, and are not tissue-selective. This project aims to determine whether ice slurry can be used as a novel, injectable, drug-free, and tissue-selective method of cryoneurolysis and resulting analgesia. METHODS: The authors developed an injectable and selective method of cryoneurolysis using biocompatible ice slurry, using rat sciatic nerve to investigate the effect of slurry injection on the structure and function of the nerve. Sixty-two naïve, male Sprague-Dawley rats were used in this study. Advanced Coherent anti-Stokes Raman Scattering microscopy, light, and fluorescent microscopy imaging were used at baseline and at various time points after treatment for evaluation and quantification of myelin sheath and axon structural integrity. Validated motor and sensory testing were used for evaluating the sciatic nerve function in response to ice slurry treatment. RESULTS: Ice slurry injection can selectively target the rat sciatic nerve. Being injectable, it can infiltrate around the nerve. The authors demonstrate that a single injection is safe and selective for reversibly disrupting the myelin sheaths and axon density, with complete structural recovery by day 112. This leads to decreased nocifensive function for up to 60 days, with complete recovery by day 112. There was up to median [interquartile range]: 68% [60 to 94%] reduction in mechanical pain response after treatment. CONCLUSIONS: Ice slurry injection selectively targets the rat sciatic nerve, causing no damage to surrounding tissue. Injection of ice slurry around the rat sciatic nerve induced decreased nociceptive response from the baseline through neural selective cryoneurolysis.

Benefit of regional anaesthesia on postoperative pain following mastectomy: the influence of catastrophising.

BACKGROUND: Previous studies suggest that truncal regional anaesthesia (TRA), including techniques such as paravertebral block, may contribute significantly to analgesia after mastectomy. However, the severity and impact of postoperative pain varies markedly amongst individuals, making the identification of patients who would benefit most from TRA a potentially important step toward personalised perioperative care. METHODS: In this prospective observational study, mastectomy patients (n=122) were recruited and systematically assessed for psychosocial characteristics including pain catastrophising before surgery, and either received preoperative TRA (n=57) or no block (n=65). RESULTS: Age, baseline pain, and psychosocial traits did not differ between these groups. TRA was associated with lower overall pain scores and opioid consumption perioperatively, with a larger proportion of patients without block (50% vs 28%) reporting moderate-severe pain (more than three/10) on the day of surgery. Mixed model analysis of variance revealed a significant interaction between catastrophising and TRA, such that amongst patients with high baseline catastrophising, TRA was associated with substantially lower pain severity score (58% lower), while amongst patients with low baseline catastrophising, TRA was associated with only 18% lower pain severity. At 2 weeks, this interaction between baseline catastrophising and TRA was also observed when examining surgical pain burden, with higher baseline catastrophising patients who had received TRA reporting lower pain and less frequent opioid use (40% vs 70% of patients). CONCLUSIONS: TRA provided immediate analgesic benefit for patients undergoing mastectomy on the day of surgery, but this effect appeared more pronounced and sustained amongst patients with higher baseline catastrophising. CLINICAL TRIAL REGISTRATION: NCT02329574.

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).

The Science of Local Anesthesia: Basic Research, Clinical Application, and Future Directions.

Local anesthetics have been used clinically for more than a century, but new insights into their mechanisms of action and their interaction with biological systems continue to surprise researchers and clinicians alike. Next to their classic action on voltage-gated sodium channels, local anesthetics interact with calcium, potassium, and hyperpolarization-gated ion channels, ligand-gated channels, and G protein-coupled receptors. They activate numerous downstream pathways in neurons, and affect the structure and function of many types of membranes. Local anesthetics must traverse several tissue barriers to reach their site of action on neuronal membranes. In particular, the perineurium is a major rate-limiting step. Allergy to local anesthetics is rare, while the variation in individual patient's response to local anesthetics is probably larger than previously assumed. Several adjuncts are available to prolong sensory block, but these typically also prolong motor block. The 2 main research avenues being followed to improve action of local anesthetics are to prolong duration of block, by slow-release formulations and on-demand release, and to develop compounds and combinations that elicit a nociception-selective blockade.

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.

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.

Systemic Progesterone Administration in Early Life Alters the Hyperalgesic Responses to Surgery in the Adult: A Study on Female Rats.

BACKGROUND: There has recently been a substantial increase in the survival of prematurely born neonates and an increase of in utero surgeries. Noxious stimulation in the newborn alters the pain response to injury in adult life. Progesterone, an effective antihyperalgesic agent in the adult, is at high concentration in the pregnant mother. Therefore, we investigated the effects of early-life progesterone on postsurgical outcomes in adult rats. METHODS: Female rat pups were administered progesterone or vehicle during the first 7 days postpartum (P1-P7). A second control group had no injections. Half of each of these groups received an incision of the hindpaw at P3 and the other half did not. At P60, all groups of these now adult rats received a second paw incision. Tactile sensitivity and thermal sensitivity were measured weekly at P14-P42 (period I), at P60 (just before the second incision), and every 2 days of P61-P70 (period II). At P67, rats were fixed by systemic paraformaldehyde perfusion and their spinal cords taken for staining and immunocytochemical analysis of activated p-p38 mitogen-activated protein kinase. RESULTS: Rats with surgery at P3 had greater tactile and thermal hyperalgesia in period I than the nonoperated rats, a difference abolished by progesterone treatment. P3 incision also resulted in long-lasting tactile and thermal hyperalgesia after the P60 incision (period II), both of which were markedly smaller in degree and faster to resolve in rats receiving early progesterone. Even in rats that were not operated on in period I, neonatal progesterone lessened the tactile hyperalgesia in period II. More spinal cells showed p-p38 staining in vehicle-treated rats as a result of the early-life incision but not in those treated with progesterone. CONCLUSIONS: These findings suggest that endogenously high progesterone in utero may have a similarly protective action and that the development of nociceptive circuitry can be strongly influenced by neonatal progesterone.

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.

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.

Local pathology and systemic serum bupivacaine after subcutaneous delivery of slow-releasing bupivacaine microspheres.

BACKGROUND: Prolonged local anesthesia, particularly desirable to minimize acute and chronic postoperative pain, has been provided by microspheres that slowly release bupivacaine (MS-Bup). In this study, we report on the systemic drug concentrations and the local dermatopathology that occur after subcutaneous injection of MS-Bup. METHODS: Rats (approximately 300 g) were injected under the dorsolumbar skin with MS-Bup containing 40 mg of bupivacaine (base) or with 0.4 mL of 0.5% bupivacaine-HCl (BupHCl; 1.78 mg bupivacaine). Blood was drawn, under sevoflurane anesthesia, at 10 minutes to 144 hours, and the serum analyzed for total bupivacaine by liquid chromatography-tandem mass spectrometry. In different animals, skin punch biopsies (4 mm) were taken at 1, 3, 7, 14, and 30 days after the same drug injections, sectioned at 5 µm, and stained with hematoxylin-eosin. Samples from skin injected with BupHCl, with MS-Bup suspended in carboxymethyl cellulose (MS-Bup.CMC), or in methyl cellulose (MS-Bup.MC) were compared with their respective drug-free controls (placebos). RESULTS: Serum bupivacaine reached a maximal average value (n = 8) of 194.9 ng/mL at 8 hours after injection of MS-Bup (95% upper prediction limit = 230.2 ng/mL), compared with the maximal average (n = 6) serum level of 374.9 ng/mL (95% prediction limit = 470.6 ng/mL) at 30 minutes after injection of BupHCl. Serum bupivacaine decreased to undetectable levels (<3.23 ng/mL) at 8 hours after BupHCl and was detectable at approximately 20% of the maximal value at 144 hours after MS-Bup injection. BupHCl injection resulted in moderate lymphocytic infiltration of skeletal muscle at 1 and 3 days. MS-Bup.CMC and placebo-CMC caused extensive infiltration of macrophages, lymphocytes, and some neutrophils at 1 to 7 days, whereas MS-Bup.MC and placebo-MC caused only mild inflammation. CONCLUSIONS: Subcutaneous administration of microspheres releasing bupivacaine results in lower blood levels lasting for much longer times than those from bupivacaine solution.

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.

Kinetics of uptake and washout of lidocaine in rat sciatic nerve in vitro.

BACKGROUND: The potency and efficacy of local anesthetics injected clinically for peripheral nerve block depends strongly on the rate of neural drug uptake. However, because diffusion into surrounding tissues and removal by the vascular system are major factors in the overall distribution of lidocaine in vivo, true kinetics of drug/neural tissue interactions must be studied in the absence of those confounding factors. METHODS: Uptake: Ensheathed or desheathed isolated rat sciatic nerves were exposed to [(14)C]-lidocaine for 0 to 180 minutes and then removed and the lidocaine content of nerve and sheath analyzed. Washout: Isolated nerves were soaked in [(14)C]-lidocaine for 60 minutes and then placed in lidocaine-free solution for 0 to 30 minutes, with samples removed at different times to assess the drug content. Experimental variables included the effects of the ensheathing epineurium, lidocaine concentration, pH, presence of CO(2)-bicarbonate, and incubation duration. RESULTS: The equilibrium uptake of lidocaine increased with incubation time, concentration, and the fraction of molecules in the nonionized form. The uptake rate was unaffected by drug concentration, but was about halved by the presence of the epineurial sheath, with the washout rate slowed less. Slight alkalinization, from pH 6.8 to pH 7.4, by bicarbonate-CO(2) buffer or a nonbicarbonate buffer, enhanced the neural uptake, and to the same degree. The washout of lidocaine was faster after shorter incubations at high concentrations than when equal amounts of lidocaine were taken up after long incubations at low lidocaine concentrations. CONCLUSION: Lidocaine enters a nerve by a process other than free diffusion, through an epineurial sheath that is a slight obstacle. Given the rapid entry in vitro compared with the much smaller and transient content measured in vivo, it seems highly unlikely that lidocaine equilibrates with the nerve during a peripheral blockade.

Preventive analgesia by local anesthetics: the reduction of postoperative pain by peripheral nerve blocks and intravenous drugs.

The use of local anesthetics to reduce acute postoperative pain has a long history, but recent reports have not been systematically reviewed. In addition, the need to include only those clinical studies that meet minimum standards for randomization and blinding must be adhered to. In this review, we have applied stringent clinical study design standards to identify publications on the use of perioperative local anesthetics. We first examined several types of peripheral nerve blocks, covering a variety of surgical procedures, and second, we examined the effects of intentionally administered IV local anesthetic (lidocaine) for suppression of postoperative pain. Thirdly, we have examined publications in which vascular concentrations of local anesthetics were measured at different times after peripheral nerve block procedures, noting the incidence when those levels reached ones achieved during intentional IV administration. Importantly, the very large number of studies using neuraxial blockade techniques (epidural, spinal) has not been included in this review but will be dealt with separately in a later review. The overall results showed a strongly positive effect of local anesthetics, by either route, for suppressing postoperative pain scores and analgesic (opiate) consumption. In only a few situations were the effects equivocal. Enhanced effectiveness with the addition of adjuvants was not uniformly apparent. The differential benefits between drug delivery before, during, or immediately after a surgical procedure are not obvious, and a general conclusion is that the significant antihyperalgesic effects occur when the local anesthetic is present during the acute postoperative period, and its presence during surgery is not essential for this action.

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.

Shifts in cell-type expression accompany a diminishing role of spinal p38-mapkinase activation over time during prolonged postoperative pain.

BACKGROUND: Surgery often causes prolonged postoperative pain, the mechanisms of which are unknown. The authors investigated the role of p38, a pain-associated mitogen-activated protein kinase, in induction and maintenance of such pain. METHODS: Male rats were subjected to the skin-muscle incision retraction procedure at the saphenous region; the procedure causes ~4 weeks of secondary tactile hyperalgesia in the ipsilateral plantar region, indicating central sensitization. The spinal cord was sectioned from L3 and L4 + L5 vertebral segments and stained for activated p38 (P-p38) at postoperative day 3 (POD 3), just as secondary hyperalgesia develops; at PODs 10-12, the time of maximum hyperalgesia; and at POD 35, after the resolution of hyperalgesia. Some sections were costained for microglia, astrocytes, and neurons. Intrathecal injections of a P-p38 inhibitor were performed at POD 2 or POD 9, and subsequent changes in pain were monitored. RESULTS: Skin-muscle incision retraction increased the numbers of dorsal horn P-p38 positive cells in L3 by ~3-fold and in L4 + L5 by ~7-fold from POD 3 to PODs 11-12. This increase was accompanied by a shift from microglia to neurons, resulting in a ~20-fold increase in P-p38-positive neurons in L4-L5 over this time. No P-p38 was detected in astrocytes. A P-p38 inhibitor given at POD 2 prevented development of secondary hypersensitivity, but when given at POD 9 the same dose gave weak relief of pain for less than 3 h. CONCLUSIONS: Spinal P-p38 mitogen-activated protein kinase, activated after incision retraction, is important for the induction of prolonged pain, but despite increased pain near the time of maximum pain, its functional importance for the maintenance of pain is not great.

Prolonged suppression of postincisional pain by a slow-release formulation of lidocaine.

BACKGROUND: Postoperative pain can occur despite nerve blocks during the surgical period. Here we tested Xybrex (Orthocon, Inc., Irvington, NY), a slow-release formulation of lidocaine that blocks rat sciatic nerve for 1-2 days, for its ability to suppress postincisional pain. METHODS: A plantar paw incision was made in rats, either along the midline (Brennan model) or at the lateral edge, 30 min after different treatment groups received either lidocaine (0.2 ml, 2%) or Xybrex implant at the ipsilateral sciatic nerve or Xybrex at the contralateral sciatic nerve. Behavioral testing by von Frey filaments occurred at 2 and 6 h postoperatively and for the next 10 postoperative days. The fractional response (paw withdrawal responses per 10 pokes) was scored at each time. RESULTS: Mechanosensitivity from the Brennan paw incision was reduced throughout the postoperative period by ipsilateral Xybrex, although lidocaine injection almost had no effect. Contralateral Xybrex had a weaker but still significant antihyperalgesic effect, converging to that from ipsilateral Xybrex at postoperative day 2. Xybrex at the nuchal midline reduced allodynia for only postoperative days 1-3, whereas hyperalgesia was reduced continuously after postoperative day 2. Hyperalgesia from the lateral incision was also reduced by ipsilateral Xybrex but not by contralateral Xybrex. CONCLUSIONS: Implants of slow-release lidocaine formulations are most effective against postincisional pain when placed at the ipsilateral nerve innervating the area of incision. Contralateral nerve implants are somewhat less effective, probably acting by releasing lidocaine into the systemic circulation. There appears to be a differential role of central sensitization between postincisional allodynia and 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.

Endothelin-B receptor activation triggers an endogenous analgesic cascade at sites of peripheral injury.

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.