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.
Antibodies are important tools for the study of protein expression, but are often used without full validation. In this study, we use Western blots to characterize antibodies targeted to the N- (NT) or C-termini (CT) and the second (IL2) or third intracellular (IL3) loops 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 50kD 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 kD band, but failed to detect endogenous ETB in rat brain. Bands detected by the CT-targeted or IL3-targeted antibodies were found to be unrelated to ETB. Our findings show that functional ETB receptors can be detected at 50 kD or 37 kD on Western blot, with drastic differences in antibody affinity for these bands. The 37 kD band likely reflects ETB receptor processing, which appears to be dependent on cell type and/or culture condition.
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.
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 (2?M) 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 (0.2-0.4?M). 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.
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.
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.
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.
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.
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.
Resolvins, including D and E series resolvins, are endogenous lipid mediators generated during the resolution phase of acute inflammation from the omega-3 polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Resolvins have potent anti-inflammatory and pro-resolution actions in several animal models of inflammation. Recent findings also demonstrate that resolvin E1 and resolvin D1 can each potently dampen inflammatory and postoperative pain. This review focuses on the mechanisms by which resolvins act on their receptors in immune cells and neurons to normalize exaggerated pain via regulation of inflammatory mediators, transient receptor potential (TRP) ion channels, and spinal cord synaptic transmission. Resolvins may offer novel therapeutic approaches for preventing and treating pain conditions associated with inflammation.
Capsaicin selectively binds to TRPV1, the vanilloid subtype 1 of the superfamily of transient receptor potential ion channels, which is highly expressed in pain-transmitting C fibers. Recent reports have demonstrated that the coadministration of capsaicin with a local anesthetic (LA) at the rat sciatic nerve elicits a prolonged nociceptive-selective nerve block, suggesting that activation of the TRPV1 receptor may allow LAs to enter the nerve through the TRPV1 pore. In previous studies, we demonstrated that transdermal amitriptyline achieves clinical analgesic effects and is more potent than lidocaine. Here we examine whether the combined application of amitriptyline and capsaicin as a transdermal patch will produce prolonged cutaneous analgesia compared with amitriptyline alone.
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 PKC?. 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 G?q/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 G?q/11 activation.
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.
Postoperative pain slows surgical recovery, impacting the return of normal function for weeks, months, or longer. Here we report the antihyperalgesic actions of a new compound, resolvin D1 (RvD1), known to reduce inflammation and to suppress pain after peripheral nerve injury, on the acute pain occurring after paw incision and the prolonged pain after skin-muscle retraction. Injection of RvD1 (20-40ng) into the L5-L6 intrathecal space 30minutes before surgery reduces the postincisional primary mechanical hypersensitivity, lowering the peak change by approximately 70% (with 40ng) and reducing the area under the curve (AUC) for the entire 10-day postincisional course by approximately 60%. Intrathecal injection of RvD1 on postoperative day (POD) 1 reduces the hyperalgesia to the same level as that from preoperative injection within a few hours, an effect that persists for the remaining PODs. Tactile allodynia and hyperalgesia following the skin/muscle incision retraction procedure, measured at the maximum values 12 to 14days, is totally prevented by intrathecal RvD1 (40ng) given at POD 2. However, delaying the injection until POD 9 or POD 17 results in RvD1 causing only transient and incomplete reversal of hyperalgesia, lasting for <1day. These findings demonstrate the potent, effective reduction of postoperative pain by intrathecal RvD1 given before or shortly after surgery. The much more limited effect of this compound on retraction-induced pain, when given 1 to 2weeks later, suggests that the receptors or pathways for resolvins are more important in the early than the later stages of postoperative pain. Single intrathecal injections of resolvin D1 in rats before or 1 to 2days after surgery strongly reduce postoperative pain for several weeks.
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.
Nerve blocks of long duration are often desirable in perioperative and postoperative situations. The relationship between the duration of such blocks and the rate at which a local anesthetic is released is important to know for developing a localized drug delivery system that will optimize block duration.
Subcutaneous injection of endothelin-1 (ET-1) into the glabrous skin of the rats 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.
Exposure to endothelin-1 (ET-1, 50 nM) of sensory neurons, acutely isolated from rat dorsal root ganglia (DRG), results in an increase in the number of action potentials elicited by a linear ramp of stimulating current. The changes are complete in 5 min after ET-1 treatment and do not reverse in 5-10 min after ET-1s removal. Neither the resting potential, nor the threshold potential for the first or second action potentials, nor their rate-of-rise or decay, are changed by ET-1 exposure, but the slow depolarizations which occur before the first and second action potentials during the current ramp are increased by ca. 50% by ET-1. The delayed rectifier type of K(+) currents (I(K)), measured under whole-cell voltage clamp, are depressed by approximately 30% during such exposure to ET-1. The voltage-dependent gating of steady-state I(K) and the current kinetics are unchanged by ET-1, leaving the sole effect as a drop in the number of available channels. I(K) is affected by ET-1 only in Isolectin B(4)-positive cells, suggesting that there may be a selective action in enhancing impulse activity on this class of nociceptive neuron. This decrease in I(K) will potentiate the excitability-inducing actions of the previously reported negative shift in tetrodotoxin-resistant Na(+) channel gating in such neurons.
Functional blockade of peripheral nerves is the primary objective of local anesthesia, and it is often desirable to have a persistent blockade, sustained throughout and beyond a surgical procedure. Current local anesthetics give effective analgesia for <8-12 h after a single bolus injection. We report on an implantable, controlled-release drug delivery system intended for use in bone and consisting of a Food and Drug Administration-approved matrix containing lidocaine that is capable of local delivery for several days.
The endogenous endothelin (ET) peptides participate in a remarkable variety of pain-relatedprocesses. Pain that is elevated by inflammation, by skin incision, by cancer, during a Sickle Cell Disease crisis and by treatments that mimic neuropathic and inflammatory pain and are all reduced by local administration of antagonists of endothelin receptors. Many effects of endogenously released endothelin are simulated by acute, local subcutaneous administration of endothelin, which at very high concentrations causes pain and at lower concentrations sensitizes the nocifensive reactions to mechanical, thermal and chemical stimuli.
Minimally invasive applications of thermal and mechanical energy to selective areas of the human anatomy have led to significant advances in treatment of and recovery from typical surgical interventions. Image-guided focused ultrasound allows energy to be deposited deep into the tissue, completely noninvasively. There has long been interest in using this focal energy delivery to block nerve conduction for pain control and local anesthesia. In this study, we have performed an in vitro study to further extend our knowledge of this potential clinical application. The sciatic nerves from the bullfrog (Rana catesbeiana) were subjected to focused ultrasound (at frequencies of 0.661 MHz and 1.986 MHz) and to heated Ringers solution. The nerve action potential was shown to decrease in the experiments and correlated with temperature elevation measured in the nerve. The action potential recovered either completely, partially or not at all, depending on the parameters of the ultrasound exposure. The reduction of the baseline nerve temperature by circulating cooling fluid through the sonication chamber did not prevent the collapse of the nerve action potential; but higher power was required to induce the same endpoint as without cooling. These results indicate that a thermal mechanism of focused ultrasound can be used to block nerve conduction, either temporarily or permanently.
The endogenous peptide endothelin-1 (ET-1), originally identified as a potent vasoconstrictor, plays a role in a number of painful conditions. In this review article we discuss the mechanisms that are essential for local sensitization by subcutaneously administered ET-1, and report evidence of ET-1s ability to sensitize distant regions of the body, through the central nervous system and, likely, coupling through the spinal cord. In addition, we will review the latest information on the role of ET-1 in cancerous and non-cancerous conditions. Cancer pain has indeed been shown to be attenuated by antagonists of endothelin receptors, and ET-1 is known to be secreted by cancer cells of many different histologic types. Furthermore, a growing body of evidence links increased expression and secretion of ET-1 to the occurrence of non-cancer related pain syndromes, such as inflammatory and neuropathic pain syndromes.
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.
Intraplantar injection of endothelin-1 (ET-1) (1.5-10 muM) in the rat produces mechanical allodynia. Here we identify the receptor subtypes for ET-1, glutamate and CGRP critical to such allodynia. Antagonism of ET(A) or ET(B) receptors alone, by BQ123 or BQ788, respectively, only partially suppressed allodynia; the combined antagonists prevented allodynia, showing the involvement of both receptor subtypes. Co-injection of NMDA receptor antagonists, (+)MK-801 or D-AP5, with ET-1 also prevented allodynia. In contrast, co-injection of the CGRP1 antagonist CGRP(8-37) attenuated only the later phase of allodynia (>30 min). A mechanistic basis for these effects is shown by ET-1s ability to enhance basal release from cultured sensory neurons of glutamate and CGRP (2.4-fold and 5.7-fold, respectively, for 10 nM ET-1). ET(A) blockade reduced ET-1s enhancement of basal CGRP release by approximately 80%, but basal glutamate release by only approximately 30%. ET-1 also enhanced the capsaicin-stimulated release of CGRP (up to 2-fold for 0.3 nM ET-1), but did not change capsaicin-stimulated glutamate release. Release stimulated by elevated K+ was not altered by ET(A) blockade, nor did blockade of ET(B) reduce any type of release. Thus, ET-1 may induce release of glutamate and CGRP from nerve terminals innervating skin, thereby sensitizing primary afferents, accounting for ET-1-dependent tactile allodynia.
Injection of endothelin-1 (ET-1) into the plantar rat hindpaw causes acute pain at high concentrations and tactile sensitization at low concentrations. The pro-nociceptive actions are driven through ET(A) receptors for both levels of [ET-1], but the ET(B) receptors are only pro-nociceptive for allodynia from low [ET-1] and anti-nociceptive for pain from high [ET-1]. The goal of the present work was to discriminate the roles of the ET receptors in the acute hyperalgesia from inflammation by complete Freunds adjuvant (CFA, 20 mg/paw) into the rat hindpaw. Selective antagonists were injected l0 min before and then together with CFA. An ET(A) receptor antagonist, BQ-123, reduced CFA-induced thermal hyperalgesia (by up to 50%), as did an ET(B) receptor antagonist, BQ-788 (by up to 66%). BQ-123 and BQ-788 also delayed the onset (by 1.5 - 2 h) but insignificantly reduced the maximum degree of CFA-induced allodynia (~10%). Surprisingly, an ET(B) receptor agonist, IRL-1620, also reduced maximum thermal hyperalgesia induced by CFA, suppressed peak allodynia and delayed its occurrence by ~ 3 h. The latter actions of IRL-1620 were reversed by co-administration of BQ-788, naloxone hydrochloride and the peripherally restricted opiate receptor antagonist naloxone methiodide, and by antiserum against ?-endorphin. These findings demonstrate an important role for endogenous ET-1 in acute inflammatory pain and a dual action of ET(B) receptors, including a pro-algesic action along with the important activation of a local analgesic pathway, implying that at least two different ET(B) receptors contribute to modulation of inflammatory pain.
To minimize acute postoperative pain, a new formulation of slowly released bupivacaine was developed.
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