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Extracellular vesicles (EVs) theranostic potential is under intense investigation. There is a wealth of information highlighting the role that EVs and the secretome play in disease and how these are being utilized for clinical trials and novel therapeutic possibilities. However, understanding of the physiological and pathological roles of EVs remain incomplete. The challenge lies in reaching a consensus concerning standardized quality-controlled isolation, storage, and sample preparation parameters. Interest in circulating EV cargo as diagnostic and prognostic biomarkers is steadily growing. Though promising, various limitations need to be addressed before there can be successful, full-scale therapeutic use of approved EVs. These limitations include obtaining or manufacturing from the appropriate medium (e.g., from bodily fluid or cell culture), loading and isolating EVs, stability, and storage, standardization of processing, and determining potency. This review highlights specific topics, including circulation of abnormal EVs contribute to human disease and the theranostic potential of EVs. Theranostics is defined as a combination of the word's therapeutics and diagnostics and describes how a specific medicine or technique can function as both. Key findings include, (1) EVs and the secretome are future theranostics which will be utilized as both biomarkers for diagnosis and as therapeutics, (2) basic and translational research supports clinical trials utilizing EVs/secretome, and (3) additional investigation is required to fully unmask the theranostic potential of EVs/secretome in specific diseases and injuries., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Neuropathic pain is caused by the lesion or disease of the somatosensory system and can be initiated and/or maintained by both central and peripheral mechanisms. Nerve injury leads to neuronal damage and apoptosis associated with the release of an array of pathogen- or damage-associated molecular patterns to activate inflammasomes. The activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome contributes to neuropathic pain and may represent a novel target for pain therapeutic development. In the current review, we provide an up-to-date summary of the recent findings on the involvement of NLRP3 inflammasome in modulating neuropathic pain development and maintenance, focusing on peripheral neuropathic conditions. Here we provide a detailed review of the mechanisms whereby NLRP3 inflammasomes contribute to neuropathic pain via (1) neuroinflammation, (2) apoptosis, (3) pyroptosis, (4) proinflammatory cytokine release, (5) mitochondrial dysfunction, and (6) oxidative stress. We then present the current research literature reporting on the antinociceptive effects of several natural products and pharmacological interventions that target activation, expression, and/or regulation of NLRP3 inflammasome. Furthermore, we emphasize the effects of microRNAs as another regulator of NLRP3 inflammasome. In conclusion, we summarize the possible caveats and future perspectives that might provide successful therapeutic approaches against NLRP3 inflammasome for treating or preventing neuropathic pain conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Preclinical and clinical studies have reported sex differences in pain and analgesia. These differences may be linked to anatomical structures of the central nervous system pain modulatory circuitry, and/or hormonal milieu. The midbrain periaqueductal gray (PAG) is a critical brain region for descending inhibition of pain. The PAG projects to the rostral ventromedial medulla (RVM), which projects bilaterally to the spinal cord to inhibit pain. In addition to pain, this descending circuit (or pathway) can be engaged by endogenous opioids (i.e., endorphins) or exogenous opioids (i.e., morphine), and we have previously reported sex differences in the activation of this circuit during pain and analgesia. Forebrain structures, including the amygdala, project to and engage the PAG-RVM circuit during persistent inflammatory pain. However, there are limited studies in females detailing this amygdalar-PAG pathway and its involvement during persistent inflammatory pain. The objective of the present study was to delineate the neural projections from the amygdala to the PAG in male and female rats to determine if they are sexually distinct in their anatomical organization. We also examined the activation of this pathway by inflammatory pain and the co-localization of receptors for estrogen. Injection of the retrograde tracer fluorogold (FG) into the ventrolateral PAG (vlPAG) resulted in dense retrograde labeling in both the central amygdala (CeA) and medial amygdala (MeA). While the number of CeA-vlPAG neurons were comparable between the sexes, there were more MeA-vlPAG neurons in females. Inflammatory pain resulted in greater activation of the amygdala in males; however, females displayed higher Fos expression within CeA-vlPAG projection neurons. Females expressed higher ERα in the MeA and CeA and the same was true of the projection neurons. Together, these data indicate that although the MeA-vlPAG projections are denser in females, inflammatory pain does not significantly activate these projections. In contrast, inflammatory pain resulted in a greater activation of the CeA-vlPAG pathway in females. As females experience a greater number of chronic pain syndromes, the CeA-vlPAG pathway may play a facilitatory (and not inhibitory) role in pain modulation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Abstract: The role of the major estrogen estradiol (E2) on orofacial pain conditions remains controversial with studies reporting both a pronociceptive and antinociceptive role of E2. E2 modulation of peripheral serotonergic activity may be one mechanism underlying the female prevalence of orofacial pain disorders. We recently reported that female rats in proestrus and estrus exhibit greater serotonin (5HT)-evoked orofacial nocifensive behaviors compared with diestrus and male rats. Further coexpression of 5HT 2A receptor mRNA in nociceptive trigeminal sensory neurons that express transient receptor potential vanilloid 1 ion channels contributes to pain sensitization. E2 may exacerbate orofacial pain through 5HT-sensitive trigeminal nociceptors, but whether low or high E2 contributes to orofacial pain and by what mechanism remains unclear. We hypothesized that steady-state exposure to a proestrus level of E2 exacerbates 5HT-evoked orofacial nocifensive behaviors in female rats, explored the transcriptome of E2-treated female rats, and determined which E2 receptor contributes to sensitization of female trigeminal sensory neurons. We report that a diestrus level of E2 is protective against 5HT-evoked orofacial pain behaviors, which increase with increasing E2 concentrations, and that E2 differentially alters several pain genes in the trigeminal ganglia. Furthermore, E2 receptors coexpressed with 5HT 2A and transient receptor potential vanilloid 1 and enhanced capsaicin-evoked signaling in the trigeminal ganglia through estrogen receptor α. Overall, our data indicate that low, but not high, physiological levels of E2 protect against orofacial pain, and we provide evidence that estrogen receptor α receptor activation, but not others, contributes to sensitization of nociceptive signaling in trigeminal sensory neurons., (Copyright © 2022 International Association for the Study of Pain.)

Oxidative stress, resulting from an imbalance between the formation of damaging free radicals and availability of protective antioxidants, can contribute to peripheral neuropathic pain conditions. Reactive oxygen and nitrogen species, as well as products of the mitochondrial metabolism such as superoxide anions, hydrogen peroxide, and hydroxyl radicals, are common free radicals. Nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is a transcription factor encoded by the NFE2L2 gene and is a member of the cap 'n' collar subfamily of basic region leucine zipper transcription factors. Under normal physiological conditions, Nrf2 remains bound to Kelch-like ECH-associated protein 1 in the cytoplasm that ultimately leads to proteasomal degradation. During peripheral neuropathy, Nrf2 can translocate to the nucleus, where it heterodimerizes with muscle aponeurosis fibromatosis proteins and binds to antioxidant response elements (AREs). It is becoming increasingly clear that the Nrf2 interaction with ARE leads to the transcription of several antioxidative enzymes that can ameliorate neuropathy and neuropathic pain in rodent models. Current evidence indicates that the antinociceptive effects of Nrf2 occur via reducing oxidative stress, neuroinflammation, and mitochondrial dysfunction. Here, we will summarize the preclinical evidence supporting the role of Nrf2 signaling pathways and Nrf2 inducers in alleviating peripheral neuropathic pain.

Orofacial pain disorders are predominately experienced by women. Progesterone, a major ovarian hormone, is neuroprotective and antinociceptive. We recently reported that progesterone attenuates estrogen-exacerbated orofacial pain behaviors, yet it remains unclear what anatomical substrate underlies progesterone's activity in the trigeminal system. Progesterone has been reported to exert protective effects through actions at intracellular progesterone receptors (iPR), membrane-progesterone receptors (mPR), or sigma 1 receptors (Sig-1R). Of these, the iPR and Sig-1R have been reported to have a role in pain. Progesterone can also have antinociceptive effects through its metabolite, allopregnanolone. Two enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase (3α-HSD), are required for the metabolism of progesterone to allopregnanolone. Both progesterone and allopregnanolone rapidly attenuate pain sensitivity, implicating action of either progesterone at Sig-1R and/or conversion to allopregnanolone which targets GABA A receptors. In the present study, we investigated whether Sig-1 Rs are expressed in nociceptors within the trigeminal ganglia of cycling female rats and whether the two enzymes required for progesterone metabolism to allopregnanolone, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are also present. Adult female rats from each stage of the estrous cycle were rapidly decapitated and the trigeminal ganglia collected. Trigeminal ganglia were processed by either fluorescent immunochemistry or western blotting to for visualization and quantification of Sig-1R, 5α-reductase, and 3α-hydroxysteroid dehydrogenase. Here we report that Sig-1Rs and both enzymes involved in progesterone metabolism are highly expressed in a variety of nociceptive sensory neuron populations in the female rat trigeminal ganglia at similar levels across the four stages of the estrous cycle. These data indicate that trigeminal sensory neurons are an anatomical substrate for the reported antinociceptive activity of progesterone via Sig-1R and/or conversion to allopregnanolone.

Orofacial pain disorders involving trigeminal sensory neurons disproportionately affect women and can be modulated by hormones, especially estrogen (E2). Proinflammatory mediators, like serotonin (5HT), can act on sensory neurons expressing the transient receptor potential vanilloid 1 (TRPV1) ion channel, resulting in peripheral sensitization. We previously reported peripheral 5HT evokes greater pain behaviors in the hindpaw of female rats during proestrus and estrus, stages when E2 fluctuates. It is unknown if this interaction is comparable in the trigeminal system. We hypothesized that E2 exacerbates 5HT-evoked nocifensive pain behaviors and pain signaling in female trigeminal sensory neurons. We report 5HT-evoked nocifensive behaviors are significantly higher during estrus and proestrus, which is attenuated by blocking the 5HT 2A receptor. The comparable dose of 5HT was not nociceptive in males unless capsaicin was also administered. When administered with capsaicin, a lower dose of 5HT evoked trigeminal pain behaviors in females during proestrus. Further, basal 5HT content in the vibrissal pad was higher in cycling females compared to males. Ex vivo , E2 enhanced 5HT-potentiated CGRP release from trigeminal neurons, which was not significantly reduced by blocking the 5HT 2A receptor. Our data indicates that estrogen fluctuation influences the pronociceptive effects of 5HT on trigeminal sensory neurons., Competing Interests: This work was supported by the National Institute of Dental & Craniofacial Research of the National Institutes of Health (grant number R15DE025970) and Texas Woman’s University. The authors have no conflict of interest to declare., (© 2021 The Author(s).)

Serotonin (5-hydroxytryptamine, 5HT) is a neurotransmitter and proinflammatory mediator found largely in the peripheral nervous system where it can initiate pain signaling. 5HT binds a variety of 5HT receptors on sensory nerve endings specialized in detecting noxious stimuli, termed nociceptors. A subset of sensory neurons involved in pain signaling express the transient receptor potential vanilloid 1 ion channel (TRPV1), a pain generator. 5HT can both directly activate sensory neurons and sensitize TRPV1 leading to enhanced nociceptor sensitivity (peripheral sensitization). Previous studies in male rats reported that the 5HT receptor subtype 3A (5HT3A) and TRPV1 are co-expressed on sensory neurons, but it is unknown if 5HT3A and TRPV1 are co-expressed on female sensory neurons. Given that craniofacial pain disorders occur at a 2-3x greater prevalence in women, examining pain mechanisms in female trigeminal sensory neurons that innervate the craniofacial region is critical to advancing craniofacial pain management in women. Here we examined whether (1) 5HT acting via the 5HT3A receptor produces sexually dimorphic orofacial pain behaviors in rats and (2) whether 5HT3A receptor mRNA is expressed in trigeminal sensory neurons, including the TRPV1-expressing subpopulation, and increase pain signaling. We report that 5HT evokes pain behaviors in male and female rats, which was not significantly reduced by antagonism of 5HT3A. We performed in situ hybridization to label 5HT3A and TRPV1 mRNA in trigeminal sensory neurons and found distinct cell populations with either 5HT3A mRNA or TRPV1 mRNA in males and females. Further, 5HT3A antagonism failed to reduce pain signaling in cultured trigeminal sensory neurons. These data suggest that the 5HT3A subtype on trigeminal sensory neurons innervating the orofacial soft tissues does not play a significant role in sexually dimorphic craniofacial pain disorders. As previous studies have reported that granisetron reduces masseter muscle pain, 5HT3 may play a role in sex differences in myofascial pain disorders but not in other craniofacial pain disorders.

Temporomandibular joint disorder (TMD) is associated with pain in the joint (temporomandibular joint, TMJ) and muscles involved in mastication. TMD pain dissipates following menopause but returns in some women undergoing estrogen replacement therapy. Progesterone has both anti-inflammatory and antinociceptive properties, while estrogen's effects on nociception are variable and highly dependent on both natural hormone fluctuations and estrogen dosage during pharmacological treatments, with high doses increasing pain. Allopregnanolone, a progesterone metabolite and positive allosteric modulator of the GABA A receptor, also has antinociceptive properties. While progesterone and allopregnanolone are antinociceptive, their effect on estrogen-exacerbated TMD pain has not been determined. We hypothesized that removing the source of endogenous ovarian hormones would reduce inflammatory allodynia in the TMJ of rats and both progesterone and allopregnanolone would attenuate the estrogen-provoked return of allodynia. Baseline mechanical sensitivity was measured in female Sprague-Dawley rats (150-175 g) using the von Frey filament method followed by a unilateral injection of complete Freund's adjuvant (CFA) into the TMJ. Mechanical allodynia was confirmed 24 h later; then rats were ovariectomized or received sham surgery. Two weeks later, allodynia was reassessed and rats received one of the following subcutaneous hormone treatments over 5 days: a daily pharmacological dose of estradiol benzoate (E2; 50 μg/kg), daily E2 and pharmacological to sub-physiological doses of progesterone (P4; 16 mg/kg, 16 μg/kg, or 16 ng/kg), E2 daily and interrupted P4 given every other day, daily P4, or daily vehicle control. A separate group of animals received allopregnanolone (0.16 mg/kg) instead of P4. Allodynia was reassessed 1 h following injections. Here, we report that CFA-evoked mechanical allodynia was attenuated following ovariectomy and daily high E2 treatment triggered the return of allodynia, which was rapidly attenuated when P4 was also administered either daily or every other day. Allopregnanolone treatment, whether daily or every other day, also attenuated estrogen-exacerbated allodynia within 1 h of treatment, but only on the first treatment day. These data indicate that when gonadal hormone levels have diminished, treatment with a lower dose of progesterone may be effective at rapidly reducing the estrogen-evoked recurrence of inflammatory mechanical allodynia in the TMJ., (Copyright © 2020 Hornung, Benton, Tongkhuya, Uphouse, Kramer and Averitt.)

Recent studies have reported that the transient receptor potential V1 ion channel (TRPV1), a pain generator on sensory neurons, is activated and potentiated by NADPH oxidase-generated reactive oxygen species (ROS). ROS are increased by advanced oxidation protein products (AOPPs), which activate NADPH oxidase by upregulating Nox4 expression. Our previous studies reported that Euphorbia bicolor ( Euphorbiaceae ) latex extract induced peripheral analgesia, partly via TRPV1, in hindpaw-inflamed male and female rats. The present study reports that E . bicolor latex extract also can evoke analgesia via reduction of oxidative stress biomarkers and proinflammatory cytokines/chemokines in a rat model of orofacial pain. Male and female rats were injected with complete Freund's adjuvant (CFA) into the left vibrissal pad to induce orofacial inflammation, and mechanical allodynia was measured by the von Frey method. Twenty-four hours later, rats received one injection of E. bicolor latex extract or vehicle into the inflamed vibrissal pad. Mechanical sensitivity was reassessed at 1, 6, 24, and/or 72 hours. Trigeminal ganglia and trunk blood were collected at each time point. In the trigeminal ganglia, ROS were quantified using 2',7'-dichlorodihydrofluorescein diacetate dye, Nox4 protein was quantified by Western blots, and cytokines/chemokines were quantified using a cytokine array. AOPPs were quantified in trunk blood using a spectrophotometric assay. E. bicolor latex extract significantly reduced orofacial mechanical allodynia in male and female rats at 24 and 72 hours, respectively. ROS, Nox4, and proinflammatory cytokines/chemokines were significantly reduced in the trigeminal ganglia, and plasma AOPP was significantly reduced in the trunk blood of extract-treated compared to vehicle-treated rats. In vitro assays indicate that E . bicolor latex extract possessed antioxidant activities by scavenging free radicals. Together our data indicate that the phytochemicals in E. bicolor latex may serve as novel therapeutics for treating oxidative stress-induced pain conditions., Competing Interests: The authors have no conflict of interest., (Copyright © 2019 Paramita Basu et al.)

The negative side effects of opioid-based narcotics underscore the search for alternative non-opioid bioactive compounds that act on the peripheral nervous system to avoid central nervous system-mediated side effects. The transient receptor potential V1 ion channel (TRPV1) is a peripheral pain generator activated and sensitized by heat, capsaicin, and a variety of endogenous ligands. TRPV1 contributes to peripheral sensitization and hyperalgesia, in part, via triggering the release of proinflammatory peptides, such as calcitonin gene-related peptide (CGRP), both locally and at the dorsal horn of the spinal cord. Ultrapotent exogenous TRPV1 agonists, such as resiniferatoxin identified in the latex of the exotic Euphorbia resinifera , trigger hyperalgesia followed by long lasting, peripheral analgesia. The present study reports on the analgesic properties of Euphorbia bicolor , a relative of E. resinifera , native to the Southern United States. The study hypothesized that E. bicolor latex extract induces long-lasting, non-opioid peripheral analgesia in a rat model of inflammatory pain. Both inflamed and non-inflamed adult male and female rats were injected with the methanolic extract of E. bicolor latex into the hindpaw and changes in pain behaviors were reassessed at various time points up to 4 weeks. Primary sensory neuron cultures also were treated with the latex extract or vehicle for 15 min followed by stimulation with the TRPV1 agonist capsaicin. Results showed that E. bicolor latex extract evoked significant pain behaviors in both male and female rats at 20 min post-injection and lasting around 1-2 h. At 6 h post-injection, analgesia was observed in male rats that lasted up to 4 weeks, whereas in females the onset of analgesia was delayed to 72 h post-injection. In sensory neurons, latex extract significantly reduced capsaicin-evoked CGRP release. Blocking TRPV1, but not opioid receptors, attenuated the onset of analgesia and capsaicin-induced CGRP release. Latex was analyzed by mass spectrometry and eleven candidate compounds were identified and reported here. These findings indicate that phytochemicals in the E. bicolor latex induce hyperalgesia followed by peripheral, non-opioid analgesia in both male and female rats, which occurs in part via TRPV1 and may provide novel, non-opioid peripheral analgesics that warrant further examination.

Morphine remains one of the most widely prescribed opioids for alleviation of persistent and/or severe pain; however, multiple preclinical and clinical studies report that morphine is less efficacious in females compared to males. Morphine primarily binds to the mu opioid receptor, a prototypical G-protein coupled receptor densely localized in the midbrain periaqueductal gray. Anatomical and physiological studies conducted in the 1960s identified the periaqueductal gray, and its descending projections to the rostral ventromedial medulla and spinal cord, as an essential descending inhibitory circuit mediating opioid-based analgesia. Remarkably, the majority of studies published over the following 30 years were conducted in males with the implicit assumption that the anatomical and physiological characteristics of this descending inhibitory circuit were comparable in females; not surprisingly, this is not the case. Several factors have since been identified as contributing to the dimorphic effects of opioids, including sex differences in the neuroanatomical and neurophysiological characteristics of the descending inhibitory circuit and its modulation by gonadal steroids. Recent data also implicate sex differences in opioid metabolism and neuroimmune signaling as additional contributing factors. Here we cohesively present these lines of evidence demonstrating a neural basis for sex differences in opioid modulation of pain, with a focus on the PAG as a sexually dimorphic core of descending opioid-induced inhibition and argue for the development of sex-specific pain therapeutics.

Many persistent pain conditions occur predominantly in women making pain a major women's health issue. One theory for the prevalence in females is hormone modulation of pain mechanisms. The peripheral release of the neurotransmitter serotonin (5HT) has been implicated in various sexually dimorphic pain conditions; yet no studies have examined the effect of ovarian hormones on peripheral 5HT-evoked pain behaviors. We hypothesized that peripheral 5HT evokes greater pain behaviors in female rodents during estrus and/or proestrus, stages of the estrous cycle where ovarian hormones are greatly fluctuating. Female Sprague-Dawley rats (250-350 g) from each stage of the estrous cycle, ovariectomized females, and intact males received an intraplantar hindpaw injection of 5HT (2 μg/100 μL) or saline (n = 6 per group) and thermal hyperalgesia, mechanical allodynia, or edema was measured at 0, 10, 20 and 30 min post-injection. A separate group of rats received an ipsilateral injection of the selective 5HT 2A antagonist, M100907, 15 min prior to 5HT injection. We report that females in proestrus and estrus exhibited significantly greater and/or longer lasting pain behaviors compared to males, females in diestrus, and ovariectomized females. There were no significant sex differences or estrous cycle effects on 5HT-evoked edema or 5HT content in inflamed hindpaws. Local pretreatment with the 5HT 2A receptor antagonist blocked 5HT-evoked thermal hyperalgesia and edema. These data provide evidence of a modulatory role of hormones on peripheral 5HT-evoked pain occurring via the 5HT 2A receptor., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Objective: Opioid-based analgesics are a major component of the lengthy pain management of burn patients, including military service members, but are problematic due to central nervous system-mediated side effects. Peripheral analgesia via targeted ablation of nociceptive nerve endings that express the transient receptor potential vanilloid channel 1 (TRPV1) may provide an improved approach. We hypothesized that local injection of the TRPV1 agonist resiniferatoxin (RTX) would produce long-lasting analgesia in a rat model of pain associated with burn injury., Methods: Baseline sensitivities to thermal and mechanical stimuli were measured in male and female Sprague-Dawley rats. Under anesthesia, a 100 °C metal probe was placed on the right hind paw for 30 seconds, and sensitivity was reassessed 72 hours following injury. Rats received RTX (0.25 μg/100 μL; ipl) into the injured hind paw, and sensitivity was reassessed across three weeks. Tissues were collected from a separate group of rats at 24 hours and/or one week post-RTX for pathological analyses of the injured hind paw, dorsal spinal cord c-Fos, and primary afferent neuropeptide immunoreactivity., Results: Local RTX reversed burn pain behaviors within 24 hours, which lasted through recovery at three weeks. At one week following RTX, decreased c-Fos and primary afferent neuropeptide immunoreactivities were observed in the dorsal horn, while plantar burn pathology was unaltered., Conclusions: These results indicate that local RTX induces long-lasting analgesia in a rat model of pain associated with burn. While opioids are undesirable in trauma patients due to side effects, RTX may provide valuable long-term, nonopioid analgesia for burn patients., (2016 American Academy of Pain Medicine. This work is written by US Government employees and is in the public domain in the US.)

Thermal burns among individuals working in highly stressful environments, such as firefighters and military Service Members, are common. Evidence suggests that pre-injury stress may exaggerate pain following thermal injury; however current animal models of burn have not evaluated the potential influence of pre-burn stress. This sham-controlled study evaluated the influence of prior stress exposure on post-burn thermal and mechanical sensitivity in male Sprague-Dawley rats. Rats were exposed to 20 min of inescapable swim stress or sham stress once per day for three days. Exposure to inescapable swim stress (1) increased the intensity and duration of thermal hyperalgesia after subsequent burn and (2) accelerated the onset of thermal hyperalgesia and mechanical allodynia after subsequent burn. This stress-induced exacerbation of pain sensitivity was reversed by pretreatment and concurrent treatment with the serotonin-norepinephrine reuptake inhibitor (SNRI) duloxetine. These data suggest a better understanding of mechanisms by which prior stress augments pain after thermal burn may lead to improved pain treatments for burn survivors., (Copyright © 2015 Elsevier Ltd and ISBI. All rights reserved.)

Background: Neuropathic pain is evasive to treat once developed, however evidence suggests that local administration of anesthetics near the time of injury reduces the development of neuropathic pain. As abnormal electrical signaling in the damaged nerve contributes to the initiation and maintenance of neuropathic pain, local administration of anesthetics prior to injury may reduce its development. We hypothesized that local treatment with bupivacaine prior to nerve injury in a rat model of spinal nerve ligation (SNL) would attenuate the initiation and/or maintenance of neuropathic pain behaviors., Methods: On the day prior to SNL, baseline measures of pre-injury mechanical, thermal, and/or cold sensitivity were recorded in adult male Sprague-Dawley rats. Immediately prior to SNL or sham treatment, the right L5 nerve was perineurally bathed in either 0.05 mL bupivacaine (0.5 %) or sterile saline (0.9 %) for 30 min. Mechanical allodynia, thermal hyperalgesia, and/or cold allodynia were then examined at 3, 7, 10, 14 and 21 days following SNL., Results: Rats exhibited both mechanical and cold allodynia, but not thermal hyperalgesia, within 3 days and up to 21 days post-SNL. No significant pain behaviors were observed in sham controls. Preemptive local bupivacaine significantly attenuated both mechanical and cold allodynia as early as 10 days following SNL compared to saline controls and were not significantly different from sham controls., Conclusions: These data indicate that local treatment with bupivacaine prior to surgical manipulations that are known to cause nerve damage may protect against the maintenance of chronic neuropathic pain.