Introduction In modern warfare, blast and burn insults account for over half of combat injuries. Burn-injured Service Members represent one military patient population in need of a pain management regimen that involves optimal acute pain control, reduced chronic pain, and reduced risk of addiction. Burn pain is a significant clinical problem. The current standard of care can result in decreasing analgesia or the need for dosage escalation that incurs an increased risk of significant side effects. This review describes the current understanding of many important structures and mechanisms associated with burn pain, and these subjects provide promising targets for novel analgesics. Materials and Methods An independent literature retrieval was performed by all authors using a combination of search terms, including “burn” and “pain,” alongside “glia,” “receptors,” “brain,” “allodynia,” “hyperalgesia,” “central sensitization,” “inflammation,” and “tolerance.” The search encompassed renowned reference repositories, namely PubMed (MEDLINE) and Scopus. Results Burn injuries are accompanied by complex pain originating after thermal insult, and this pain persists through treatment procedures like debridement, wound dressing, grafting, and wound closure. Understanding the molecular mechanisms that persist following burn injury is critical. This review highlights specific topics, including receptors and brain anatomy pertinent to pain, burn-induced allodynia and hyperalgesia, central sensitization, molecular signatures of burn-induced nociception and pain, and opioid analgesic tolerance. Conclusions Interventions to accelerate burn injury healing and/or diminish the deleterious effects of dysregulated inflammatory and immune responses following severe burn are needed. Therefore, elucidating the underlying molecular mechanisms of burn-injury induced pain is of the utmost importance.

Background Approximately 40% of Service Members deployed in support of Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF) and an astounding 80% of Veterans overall report experiencing pain. Currently, drugs that adequately treat pain may result in addiction and substance abuse or negative side effects such as nausea, vomiting, renal and cardiovascular issues, among other physiological and cognitive problems. Inadequate acute pain management can lead to the development of chronic pain. Combat and non-combat injuries, acute and chronic pain all have the potential to impact return-to-duty rates/decisions, thereby negatively affecting the Fighting Force. To develop more effective pain therapeutics, the molecular mechanisms contributing to the development of neuropathic pain are under intense investigation and further research is needed to fully understand neuropathic pain induction and maintenance. The overarching objective of this study is to identify microRNA (miRNA) changes in key brain regions during the onset and progression of neuropathic pain in a rodent model. Results Changes in miRNA expression were observed at day 15 post-SNL in the amygdala and thalamus. The majority of changes were observed in the left side of the brain, contralateral to the right-sided SNL injury. The DE miRNAs identified mainly in the amygdala and thalamus did not overlap between brain regions. The altered miRNAs regulate key signaling pathways and genes important in pain development. Discussion The majority of epigenetic studies investigating altered miRNA expression in the pain field have explored the peripheral nervous system. Very few studies have evaluated miRNA dynamics in the brain following neuropathic pain development. This study provides key insights into changes occurring in the brain following peripheral nerve injury. Our lab has previously identified circulating extracellular vesicle (EV) miRNAs that are altered in the blood post-SNL. There is some overlap between the blood and brain miRNAs that may serve as key biomarkers in prognosis and/or diagnosis of a peripheral nerve injury and the development of chronic pain.

Using a model of combat and operational stress reaction (COSR), our lab recently showed that exposure to an unpredictable combat stress (UPCS) procedure prior to a thermal injury increases pain sensitivity in male rats. Additionally, our lab has recently shown that circulating extracellular vesicle-microRNAs (EV-miRNAs), which normally function to suppress inflammation, were down-regulated in a male rat model of neuropathic pain. In this current study, male and female rats ex-posed to UPCS, followed by thermal injury, were evaluated for changes in circulating EV-miRNAs. Adult female and male Sprague Dawley rats were exposed to a UPCS procedure for either 2 or 4 weeks. Groups consisted of the following: nonstress (NS), stress (S), NS + thermal injury (TI), and S + TI. Mechanical sensitivity was measured, and plasma was collected at baseline, throughout the UPCS exposure, and post-thermal injury. EV-miRNA isolation was performed, followed by small RNA sequencing and subsequent data analysis. UPCS exposure alone resulted in mechanical allodynia in both male and female rats at specific time points. Thermal-injury induction occurring at peak UPCS resulted in increased mechanical allodynia in the injured hind paw compared to thermal injury alone. Differential expression of the EV-miRNAs was observed between the NS and S groups as well as between NS + TI and S + TI groups. Consistent differences in EV-miRNAs are detectable in both COSR as well as during the development of mechanical sensitivity and potentially serve as key regulators, biomarkers, and targets in the treatment of COSR and thermal-injury induced mechanical sensitivity. Perspective: This article presents the effects of unpredictable combat stress and thermal injury on EV-contained microRNAs in an animal model. These same mechanisms may exist in clinical patients and could be future prognostic and diagnostic biomarkers.(R) 2023 Published by Elsevier Inc. on behalf of United States Association for the Study of Pain, Inc All rights reserved.

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.

In the military, constant physiological and psychological stress encountered by Soldiers can lead to development of the combat and operational stress reaction (COSR), which can effect pain management. Similar effects are seen in other populations subjected to high levels of stress. Using a model of COSR, our lab recently showed that four weeks of stress prior to an injury increases pain sensitivity in male rats. With the roles of women in the military expanding and recent studies indicating sex differences in stress and pain processing, this study sought to investigate how different amounts of prior stress exposure affects thermal injury-induced mechanosensitivity in a female rat model of COSR. Adult female Sprague Dawley rats were exposed to the unpredictable combat stress (UPCS) procedure for either 2 or 4 weeks. The UPCS procedure included exposure to one stressor each day for four days. The stressors include: (1) sound stress for 30 min, (2) restraint stress for 4 h, (3) cold stress for 4 h, and (4) forced swim stress for 15 min. The order of stressors was randomized weekly. Mechanical and thermal sensitivity was tested twice weekly. After the UPCS procedure, a sub-set of rats received a thermal injury while under anesthesia. The development of mechanical allodynia and thermal hyperalgesia was examined for 14 days post-burn. UPCS exposure increased mechanosensitivity after two weeks. Interestingly, with more stress exposure, females seemed to habituate to the stress, causing the stress-induced changes in mechanosensitivity to decrease by week three of UPCS. If thermal injury induction occurred during peak stress-induced mechanosensitivity, after two weeks, this resulted in increased mechanical allodynia in the injured hind paw compared to thermal injury alone. This data indicates a susceptibility to increased nociceptive sensitization when injury is sustained at peak stress reactivity. Additionally, this data indicates a sex difference in the timing of peak stress. Post-mortem examination of the prefrontal cortex (PFC) showed altered expression of p-TrkB in 4-week stressed animals given a thermal injury, suggesting a compensatory mechanism. Future work will examine treatment options for preventing stress-induced pain to maintain the effectiveness and readiness of the Warfighter.

Diagnostic and prognostic biomarkers of nerve injury and/or pain as well as improved pain therapeutics are needed, both on the battlefield to treat injured Service Members and in the civilian sector. Our previous research indicates that there are several differentially expressed (DE) extracellular vesicle-derived microRNAs (EV-miRNAs) isolated from rat plasma following spinal nerve ligation (SNL). As such, EV-miRNAs hold promise as biomarkers and therapeutic targets. The secretome contains biological mediators, including EVs, which are released into the extracellular space. In this study we focus on evaluating EV-non-coding RNAs (ncRNAs), examine effects of SNL on key protein expression in the prefrontal cortex (PFC), and test the secretome's analgesic properties. To accomplish these goals, anesthetized male Sprague Dawley rats underwent SNL and nociceptive behavior measurements, plasma collection followed by EV RNA isolation, small RNA sequencing, and analysis. Expression of several key proteins in the PFC was determined by Wes/Jess analysis. The secretome bath was applied directly to the ligated nerve and the paw withdrawal threshold (PWT) was measured. We identified differences in several classes of ncRNAs such as piRNAs, snoRNAs, and snRNAs post-SNL. Levels of phosphorylated forms of P70S6K and ERK1 were increased in the dorsal PFC at 15 days post-SNL. Bath application of the secretome directly to the ligated nerve resulted in recovery of the reduced PWT (increased mechanical sensitivity) that is induced by SNL. Here, we have identified specific EV-ncRNAs that could contribute to the formation of pain. Furthermore, we have evaluated a novel product for analgesic efficacy that could function to exploit the underlying mechanisms that contribute to pain development, thus reducing acute pain. This is key in treating Service Members on the battlefield in order to prevent pain chronification.

Research into potentially novel biomarkers for chronic pain development is lacking. microRNAs (miRNAs) are attractive candidates as biomarkers due to their conservation across species, stability in liquid biopsies, and variation that corresponds to a pathologic state. miRNAs can be sorted into extracellular vesicles (EVs) within the cell and released from the site of injury. EVs transfer cargo molecules between cells thus affecting key intercellular signaling pathways. The focus of this study was to determine the plasma derived EV miRNA content in a chronic neuropathic pain rat model. This was accomplished by performing either spinal nerve ligation (SNL; n = 6) or sham (n = 6) surgery on anesthetized male Sprague-Dawley rats. Mechanosensitivity was assessed and plasma derived EV RNA was isolated at baseline (BL), day 3, and 15 postnerve injury. EV extracted small RNA was sequenced followed by differentially expressed (DE) miRNAs and gene target enrichment/signaling pathway analysis performed using R packages and TargetScan/Ingenuity pathway analysis (IPA), respectively. Seven of the DE miRNAs were validated by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR). The data indicated that SNL rats displayed a time-dependent threshold reduction in response to evoked stimuli from day 3 to day 15 postnerve injury. The data also revealed that 22 and 74 miRNAs at day 3 and 15, respectively, and 33 miRNAs at both day 3 and 15 were uniquely DE between the SNL and sham groups. The key findings from this proposal include (1) the majority of the DE EV miRNAs, which normally function to suppress inflammation, were downregulated, and (2) several of the plasma derived DE EV miRNAs reflect previously observed changes in the injured L5 nerve. The plasma derived DE EV miRNAs regulate processes important in the development and maintenance of neuropathic pain states and potentially serve as key regulators, biomarkers, and targets in the progression and treatment of chronic neuropathic pain. Perspective: This article describes the DE miRNA content of plasma derived EVs, comparing neuropathic pain to normal conditions. This data indicates that EV miRNAs may be important in nociception and may also serve as biomarkers for chronic pain. These results encourage further research on EV miRNAs in chronic neuropathic pain sufferers. (C) 2020 U.S. Association for the Study of Pain. Published by Elsevier Inc. All rights%51526-5900(20) X0009-5%202001/02%21 %1 %2%82%96% ELE\gdef\pts@issuepubyear{2020}reserved

BackgroundReports show that stressful events before injury exacerbates post-injury pain. The mechanism underlying stress-induced heightened thermal pain is unclear. Here, we examined the effects of chronic intermittent stress (CIS) on nociceptive behaviors and brain-derived nerve growth factor (BDNF) system in the prefrontal cortex (PFC) and hypothalamus of rats with and without thermal injury.ResultsUnstressed rats showed transient mechanical allodynia during stress exposure. Stressed rats with thermal injury displayed persistent exacerbated mechanical allodynia (P<0.001). Increased expression of BDNF mRNA in the PFC (P<0.05), and elevated TrkB and p-TrkB (P<0.05) protein levels in the hypothalamus were observed in stressed rats with thermal injury but not in stressed or thermally injured rats alone. Furthermore, administration of CTX-B significantly reduced stress-induced exacerbated mechanical allodynia in thermally injured rats (P<0.001).ConclusionThese results indicate that BDNF-TrkB signaling in PFC and hypothalamus contributes to CIS-induced exacerbated mechanical allodynia in thermal injury state.

Chronic pain afflicts roughly 44% of Service Members returning from Iraq and Afghanistan, and it is estimated that 100 million Americans suffer from this condition. The underlying molecular mechanisms of chronic pain development have remained elusive. The broad objective of this study is to identify molecular signals and indications of chronic pain in key brain regions during the onset and progression of neuropathic pain, in a rodent model. For this experiment, we collected tissue from five brain regions (thalamus, amygdala, cingulate cortex, insular cortex, and somatosensory cortex), as well as the L4–L6 spinal cord and L5 dorsal root ganglia, from rats subjected to the spared nerve injury (SNI) protocol, a model for chronic neuropathic pain. Tissue was collected at 1, 3, 7, 14 and 21 days post‐injury from each tissue and RNA extracted for mRNA sequencing (TruSeq total RNA protocol and Illumina HISCAN‐SQ platform). We present here the detailed mRNA sequencing workflow (pipeline) customized to provide the full complement of mRNA data (transcriptome) for multiple brain regions over the time course of the SNI protocol. In our initial analysis we focused on the assessment of the transcriptome of the thalamus, which is an important center of pain processing in the brain. We focused in particular on the 21 day time point, which is when the mechanical allodynia reaches its peak and therefore the most severe alterations in transcription are expected. In rats experiencing chronic pain relative to controls, we observed a reduction in the expression of several neurotransmitter receptors, such as CHRM4, CHRNB3, CHRNA5, CHRNB4, CNR1, ADORA2A, GPR85, BDKRB2, MC5R, DRD1, DRD2, and associated G proteins, such as GPR85, GNAT2, GNB3, GNG7, as well as the neurotransmitter TAC1. These findings suggest a global desensitization of neural signaling in the thalamus corresponding to chronic pain. Analysis of all time points and brain regions is underway and will provide a comprehensive picture of gene expression changes during the onset and progression of chronic pain.Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.Support or Funding InformationThis work was supported by funding from the United States Army Medical Research and Material Command.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sound stress (SS) elicits behavioral changes, including pain behaviors. However, the neuronal mechanisms underlying SS-induced pain behaviors remain to be explored. The current study examined the effects of SS on nociceptive behaviors and changes in expression of the spinal corticotropin-releasing factor (CRF) system in male Sprague Dawley rats with and without thermal pain. We also studied the effects of SS on plasma corticosterone and fecal output. Rats were exposed to 3 days of SS protocol (n = 12/group). Changes in nociceptive behaviors were assessed using thermal and mechanical pain tests. Following the induction of SS, a subgroup of rats (n = 6/group) was inflicted with thermal injury and on day 14 postburn nociceptive behaviors were reassessed. Spinal CRF receptor mRNA expression was analyzed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). In addition, plasma corticosterone and spinal CRF concentrations were quantified using enzyme-linked immunosorbent assay (ELISA). Increased defecation was observed in SS rats. SS produced transient mechanical allodynia in naive rats, whereas it exacerbated thermal pain in thermally injured rats. Spinal CRFR2 mRNA expression was unaffected by stress or thermal injury alone, but their combined effect significantly increased its expression. SS had no effect on plasma corticosterone and spinal CRF protein in postburn rats. To conclude, SS is capable of exacerbating postburn thermal pain, which is linked to increased CRFR2 gene expression in the spinal cord. Future studies have to delineate whether attenuation of CRFR2 signaling at the spinal level prevents stress-induced exacerbation of burn pain.

Changes in ion channel expression are implicated in the etiology of epilepsy. However, the molecular leading to long-term aberrant expression of ion channels are not well understood. The mechanistic/mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that mediates activity-dependent protein synthesis in neurons. mTOR is overactive in epilepsy, suggesting that excessive protein synthesis may contribute to the neuronal pathology. In contrast, we found that mTOR activity and the microRNA miR-129-5p reduce the expression of the voltage-gated potassium channel Kv1.1 in an animal model of temporal lobe epilepsy (TLE). When mTOR activity is low, Kv1.1 expression is high and the frequency of behavioral seizures is low. However, as behavioral seizure activity rises, mTOR activity increases and Kv1.1 protein levels drop. In CA1 pyramidal neurons, the reduction in Kv1.1 lowers the threshold for action potential firing. Interestingly, blocking mTOR activity with rapamycin reduces behavioral seizures and temporarily keeps Kv1.1 levels elevated. Overtime, seizure activity increases and Kv1.1 protein decreases in all animals, even those treated with rapamycin. Notably, the concentration of miR-129-5p, the negative regulator of Kv1.1 mRNA translation, increases by 21 days post-status epilepticus (SE), sustaining Kv1.1 mRNA translational repression. Our results suggest that following kainic-acid induced status epilepticus there are two phases of Kv1.1 repression: (1) an initial mTOR-dependent repression of Kv1.1 that is followed by (2) a miR-129-5p persistent reduction of Kv1.1.

The fate of a memory, whether stored or forgotten, is determined by the ability of an active or tagged synapse to undergo changes in synaptic efficacy requiring protein synthesis of plasticity-related proteins. A synapse can be tagged, but without the "capture" of plasticity-related proteins, it will not undergo long lasting forms of plasticity (synaptic tagging and capture hypothesis). What the "tag" is and how plasticity-related proteins are captured at tagged synapses are unknown. Ca(2+)/calmodulin-dependent protein kinase II α (CaMKIIα) is critical in learning and memory and is synthesized locally in neuronal dendrites. The mechanistic (mammalian) target of rapamycin (mTOR) is a protein kinase that increases CaMKIIα protein expression; however, the mechanism and site of dendritic expression are unknown. Herein, we show that mTOR activity mediates the branch-specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. mTOR inhibition decreased the dendritic levels of CaMKIIα protein and mRNA by shortening its poly(A) tail. Overexpression of the RNA-stabilizing protein HuD increased CaMKIIα protein levels and preserved its selective expression in one daughter branch over the other when mTOR was inhibited. Unexpectedly, deleting the third RNA recognition motif of HuD, the domain that binds the poly(A) tail, eliminated the branch-specific expression of CaMKIIα when mTOR was active. These results provide a model for one molecular mechanism that may underlie the synaptic tagging and capture hypothesis where mTOR is the tag, preventing deadenylation of CaMKIIα mRNA, whereas HuD captures and promotes its expression in a branch-specific manner.

Little is known about how a neuron undergoes site-specific changes in intrinsic excitability during neuronal activity. We provide evidence for a novel mechanism for mTORC1 kinase-dependent translational regulation of the voltage-gated potassium channel Kv1.1 messenger RNA (mRNA). We identified a microRNA, miR-129, that repressed Kv1.1 mRNA translation when mTORC1 was active. When mTORC1 was inactive, we found that the RNA-binding protein, HuD, bound to Kv1.1 mRNA and promoted its translation. Unexpectedly, inhibition of mTORC1 activity did not alter levels of miR-129 and HuD to favor binding to Kv1.1 mRNA. However, reduced mTORC1 signaling caused the degradation of high affinity HuD target mRNAs, freeing HuD to bind Kv1.1 mRNA. Hence, mTORC1 activity regulation of mRNA stability and high affinity HuD-target mRNA degradation mediates the bidirectional expression of dendritic Kv1.1 ion channels.