Responsiveness of multiple patient-reported outcome measures for acute postsurgical pain: primary results from the international multi-centre PROMPT NIT-1 study.

BACKGROUND: Postsurgical outcome measures are crucial to define the efficacy of perioperative pain management; however, it is unclear which are most appropriate. We conducted a prospective study aiming to assess sensitivity-to-change of patient-reported outcome measures assessing the core outcome set of domains pain intensity (at rest/during activity), physical function, adverse events, and self-efficacy. METHODS: Patient-reported outcome measures were assessed preoperatively, on day 1 (d1), d3, and d7 after four surgical procedures (total knee replacement, breast surgery, endometriosis-related surgery, and sternotomy). Primary outcomes were sensitivity-to-change of patient-reported outcome measures analysed by correlating their changes (d1-d3) with patients' global impression of change and patients' specific impression of change items as anchor criteria. Secondary outcomes included identification of baseline and patient characteristic variables explaining variance in change for each of the scales and descriptive analysis of various patient-reported outcome measures from different domains and after different surgeries. RESULTS: Of 3322 patients included (18 hospitals, 10 countries), data from 2661 patients were analysed. All patient-reported outcome measures improved on average over time; the median calculated sensitivity-to-change for all patient-reported outcome measures (overall surgeries) was 0.22 (range: 0.07-0.31, scale: 0-10); all changes were independent of baseline data or patient characteristics and similar between different procedures. CONCLUSIONS: Pain-related patient-reported outcome measures have low to moderate sensitivity-to-change; those showing higher sensitivity-to-change from the same domain should be considered for inclusion in a core outcome set of patient-reported outcome measures to assess the effectiveness and efficacy of perioperative pain management.

Machine learning and artificial intelligence in neuroscience: A primer for researchers.

Artificial intelligence (AI) is often used to describe the automation of complex tasks that we would attribute intelligence to. Machine learning (ML) is commonly understood as a set of methods used to develop an AI. Both have seen a recent boom in usage, both in scientific and commercial fields. For the scientific community, ML can solve bottle necks created by complex, multi-dimensional data generated, for example, by functional brain imaging or *omics approaches. ML can here identify patterns that could not have been found using traditional statistic approaches. However, ML comes with serious limitations that need to be kept in mind: their tendency to optimise solutions for the input data means it is of crucial importance to externally validate any findings before considering them more than a hypothesis. Their black-box nature implies that their decisions usually cannot be understood, which renders their use in medical decision making problematic and can lead to ethical issues. Here, we present an introduction for the curious to the field of ML/AI. We explain the principles as commonly used methods as well as recent methodological advancements before we discuss risks and what we see as future directions of the field. Finally, we show practical examples of neuroscience to illustrate the use and limitations of ML.

Spinal GABA transporter 1 contributes to evoked-pain related behavior but not resting pain after incision injury.

The inhibitory function of GABA at the spinal level and its central modulation in the brain are essential for pain perception. However, in post-surgical pain, the exact mechanism and modes of action of GABAergic transmission have been poorly studied. This work aimed to investigate GABA synthesis and uptake in the incisional pain model in a time-dependent manner. Here, we combined assays for mechanical and heat stimuli-induced withdrawal reflexes with video-based assessments and assays for non-evoked (NEP, guarding of affected hind paw) and movement-evoked (MEP, gait pattern) pain-related behaviors in a plantar incision model in male rats to phenotype the effects of the inhibition of the GABA transporter (GAT-1), using a specific antagonist (NO711). Further, we determined the expression profile of spinal dorsal horn GAT-1 and glutamate decarboxylase 65/67 (GAD65/67) by protein expression analyses at four time points post-incision. Four hours after incision, we detected an evoked pain phenotype (mechanical, heat and movement), which transiently ameliorated dose-dependently following spinal inhibition of GAT-1. However, the NEP-phenotype was not affected. Four hours after incision, GAT-1 expression was significantly increased, whereas GAD67 expression was significantly reduced. Our data suggest that GAT-1 plays a role in balancing spinal GABAergic signaling in the spinal dorsal horn shortly after incision, resulting in the evoked pain phenotype. Increased GAT-1 expression leads to increased GABA uptake from the synaptic cleft and reduces tonic GABAergic inhibition at the post-synapse. Inhibition of GAT-1 transiently reversed this imbalance and ameliorated the evoked pain phenotype.

Generation of a whole-brain hemodynamic response function and sex-specific differences in cerebral processing of mechano-sensation in mice detected by BOLD fMRI.

BOLD fMRI has become a prevalent method to study cerebral sensory processing in rodent disease models, including pain and mechanical hypersensitivity. fMRI data analysis is frequently combined with a general-linear-model (GLM) -based analysis, which uses the convolution of a hemodynamic response function (HRF) with the stimulus paradigm. However, several studies indicated that the HRF differs across species, sexes, brain structures, and experimental factors, including stimulation modalities or anesthesia, and hence might strongly affect the outcome of BOLD analyzes. While considerable work has been done in humans and rats to understand the HRF, much less is known in mice. As a prerequisite to investigate mechano-sensory processing and BOLD fMRI data in male and female mice, we (1) designed a rotating stimulator that allows application of two different mechanical modalities, including innocuous von Frey and noxious pinprick stimuli and (2) determined and statistically compared HRFs across 30 brain structures and experimental conditions, including sex and, stimulus modalities. We found that mechanical stimulation lead to brain-wide BOLD signal changes thereby allowing extraction of HRFs from multiple brain structures. However, we did not find differences in HRFs across all brain structures and experimental conditions. Hence, we computed a whole-brain mouse HRF, which is based on 88 functional scans from 30 mice. A comparison of this mouse-specific HRF with our previously reported rat-derived HRF showed significantly slower kinetics in mice. Finally, we detected pronounced differences in cerebral BOLD activation between male and female mice with mechanical stimulation, thereby exposing divergent processing of noxious and innocuous stimuli in both sexes.

Mechanisms inherent in acute-to-chronic pain after surgery - risk, diagnostic, predictive, and prognostic factors.

PURPOSE OF REVIEW: Pain is an expected consequence of a surgery, but it is far from being well controlled. One major complication of acute pain is its risk of persistency beyond healing. This so-called chronic post-surgical pain (CPSP) is defined as new or increased pain due to surgery that lasts for at least 3 months after surgery. CPSP is frequent, underlies a complex bio-psycho-social process and constitutes an important socioeconomic challenge with significant impact on patients' quality of life. Its importance has been recognized by its inclusion in the eleventh version of the ICD (International Classification of Diseases). RECENT FINDINGS: Evidence for most pharmacological and non-pharmacological interventions preventing CPSP is inconsistent. Identification of associated patient-related factors, such as psychosocial aspects, comorbidities, surgical factors, pain trajectories, or biomarkers may allow stratification and selection of treatment options based on underlying individual mechanisms. Consequently, the identification of patients at risk and implementation of individually tailored, preventive, multimodal treatment to reduce the risk of transition from acute to chronic pain is facilitated. SUMMARY: This review will give an update on current knowledge on mechanism-based risk, prognostic and predictive factors for CPSP in adults, and preventive and therapeutic approaches, and how to use them for patient stratification in the future.

Prognostic models for chronic postsurgical pain-Current developments, trends, and challenges.

PURPOSE OF REVIEW: Prognostic models for chronic postsurgical pain (CPSP) aim to predict the likelihood for development and severity of CPSP in individual patients undergoing surgical procedures. Such models might provide valuable information for healthcare providers, allowing them to identify patients at higher risk and implement targeted interventions to prevent or manage CPSP effectively. This review discusses the latest developments of prognostic models for CPSP, their challenges, limitations, and future directions. RECENT FINDINGS: Numerous studies have been conducted aiming to develop prognostic models for CPSP using various perioperative factors. These include patient-related factors like demographic variables, preexisting pain conditions, psychosocial aspects, procedure-specific characteristics, perioperative analgesic strategies, postoperative complications and, as indicated most recently, biomarkers. Model generation, however, varies and performance and accuracy differ between prognostic models for several reasons and validation of models is rather scarce. SUMMARY: Precise methodology of prognostic model development needs advancements in the field of CPSP. Development of more accurate, validated and refined models in large-scale cohorts is needed to improve reliability and applicability in clinical practice and validation studies are necessary to further refine and improve the performance of prognostic models for CPSP.

Behavioral Voluntary and Social Bioassays Enabling Identification of Complex and Sex-Dependent Pain-(-Related) Phenotypes in Rats with Bone Cancer.

Cancer-induced bone pain (CIBP) is a common and devastating symptom with limited treatment options in patients, significantly affecting their quality of life. The use of rodent models is the most common approach to uncovering the mechanisms underlying CIBP; however, the translation of results to the clinic may be hindered because the assessment of pain-related behavior is often based exclusively on reflexive-based methods, which are only partially indicative of relevant pain in patients. To improve the accuracy and strength of the preclinical, experimental model of CIBP in rodents, we used a battery of multimodal behavioral tests that were also aimed at identifying rodent-specific behavioral components by using a home-cage monitoring assay (HCM). Rats of all sexes received an injection with either heat-deactivated (sham-group) or potent mammary gland carcinoma Walker 256 cells into the tibia. By integrating multimodal datasets, we assessed pain-related behavioral trajectories of the CIBP-phenotype, including evoked and non-evoked based assays and HCM. Using principal component analysis (PCA), we discovered sex-specific differences in establishing the CIBP-phenotype, which occurred earlier (and differently) in males. Additionally, HCM phenotyping revealed the occurrence of sensory-affective states manifested by mechanical hypersensitivity in sham when housed with a tumor-bearing cagemate (CIBP) of the same sex. This multimodal battery allows for an in-depth characterization of the CIBP-phenotype under social aspects in rats. The detailed, sex-specific, and rat-specific social phenotyping of CIBP enabled by PCA provides the basis for mechanism-driven studies to ensure robustness and generalizability of results and provide information for targeted drug development in the future.

Experimenter familiarization is a crucial prerequisite for assessing behavioral outcomes and reduces stress in mice not only under chronic pain conditions.

Rodent behavior is affected by different environmental conditions. These do not only comprise experimental and housing conditions but also familiarization with the experimenter. However, specific effects on pain-related behavior and chronic pain conditions have not been examined. Therefore, we aimed to investigate the impact of different housing conditions, using individually ventilated and standard open top cages, inverted day-night cycles, and experimenter familiarization on male mice following peripheral neuropathy using the spared nerve injury (SNI) model. Using a multimodal approach, we evaluated evoked pain-related- using von Frey hair filaments, measured gait pattern with the CatWalk system, assessed anxiety- and depression-like behavior with the Elevated plus maze and tail suspension test, measured corticosterone metabolite levels in feces and utilized an integrative approach for relative-severity-assessment. Mechanical sensitivity differed between the cage systems and experimenter familiarization and was affected in both sham and SNI mice. Experimenter familiarization and an inverted day-night cycle reduced mechanical hypersensitivity in SNI and sham mice. SNI mice of the inverted day-night group displayed the slightest pronounced alterations in gait pattern in the Catwalk test. Anxiety-related behavior was only found in SNI mice of experimenter-familiarized mice compared to the sham controls. In addition, familiarization reduced the stress level measured by fecal corticosteroid metabolites caused by the pain and the behavioral tests. Although no environmental condition significantly modulated the severity in SNI mice, it influenced pain-affected phenotypes and is, therefore, crucial for designing and interpreting preclinical pain studies. Moreover, environmental conditions should be considered more in the reporting guidelines, described in more detail, and discussed as a potential influence on pain phenotypes.

Phenotype- and species-specific skin proteomic signatures for incision-induced pain in humans and mice.

BACKGROUND: Acute pain after surgery is common and often leads to chronic post-surgical pain, but neither treatment nor prevention is currently sufficient. We hypothesised that specific protein networks (protein-protein interactions) are relevant for pain after surgery in humans and mice. METHODS: Standardised surgical incisions were performed in male human volunteers and male mice. Quantitative and qualitative sensory phenotyping were combined with unbiased quantitative mass spectrometry-based proteomics and protein network theory. The primary outcomes were skin protein signature changes in humans and phenotype-specific protein-protein interaction analysis 24 h after incision. Secondary outcomes were interspecies comparison of protein regulation as well as protein-protein interactions after incision and validation of selected proteins in human skin by immunofluorescence. RESULTS: Skin biopsies in 21 human volunteers revealed 119/1569 regulated proteins 24 h after incision. Protein-protein interaction analysis delineated remarkable differences between subjects with small (low responders, n=12) and large incision-related hyperalgesic areas (high responders, n=7), a phenotype most predictive of developing chronic post-surgical pain. Whereas low responders predominantly showed an anti-inflammatory protein signature, high responders exhibited signatures associated with a distinct proteolytic environment and persistent inflammation. Compared to humans, skin biopsies in mice habored even more regulated proteins (435/1871) 24 h after incision with limited overlap between species as assessed by proteome dynamics and PPI. Immunohistochemistry confirmed the expression of high priority candidates in human skin biopsies. CONCLUSIONS: Proteome profiling of human skin after incision revealed protein-protein interactions correlated with pain and hyperalgesia, which may be of potential significance for preventing chronic post-surgical pain. Importantly, protein-protein interactions were differentially modulated in mice compared to humans opening new avenues for successful translational research.

Conditioned pain modulation and offset analgesia: Influence of sex, sex hormone levels and menstrual cycle on the magnitude and retest reliability in healthy participants.

BACKGROUND: Conditioned pain modulation (CPM) and offset analgesia quantify impairment of endogenous pain modulation, but magnitude and reliability vary broadly between studies, indicating influencing factors that are not currently controlled for. The aim of this study was to quantify magnitude and retest reliability of CPM and offset analgesia in healthy participants, while investigating the influence of sex and sex hormone levels. METHODS: Sixty-two participants (30 female) completed the study. We tested CPM (heat-cold paradigm) and offset analgesia on 6 days within two menstrual cycles (tests were performed in each phase of two subsequent menstrual cycles, with similar time points for men). RESULTS: Median offset effect was -29.4% in female and - 22.5% in male participants (as change from initial stimulus). Median early CPM effects were - 16.7% for women versus -13.3% for men. Reliability (intra-class correlation coefficient [ICC]) was similar between the main measures, offset effect (female: 0.48, male: 0.47) and early CPM effect (female: 0.49, male: 0.43). There was significant variance between individual experimental parameters within protocols but not between sexes or menstrual phases. While oestradiol and progesterone did not correlate with the magnitude of effect within sexes, we found that testosterone levels explained an estimated 5%-10% of variance within individual responses in all sexes. CONCLUSIONS: Our results show that the reliability of both CPM effect and offset analgesia was independent of sex and menstrual cycle phase. The magnitude of CPM and offset effects was weakly influenced by sex and testosterone levels, indicating an area for future research, rather than clinical significance. SIGNIFICANCE: This study investigated CPM and offset analgesia in parallel, across sexes and during two menstrual cycles while assessing the impact of sex hormones. Reliability seems to depend on experimental parameters rather than participant characteristics, while the magnitude of effect could be weakly linked to sex hormones and sex.

The "WWHow" Concept for Prospective Categorization of Post-operative Severity Assessment in Mice and Rats.

The prospective severity assessment in animal experiments in the categories' non-recovery, mild, moderate, and severe is part of each approval process and serves to estimate the harm/benefit. Harms are essential for evaluating ethical justifiability, and on the other hand, they may represent confounders and effect modifiers within an experiment. Catalogs and guidelines provide a way to assess the experimental severity prospectively but are limited in adaptation due to their nature of representing particular examples without clear explanations of the assessment strategies. To provide more flexibility for current and future practices, we developed the modular Where-What-How (WWHow) concept, which applies findings from pre-clinical studies using surgical-induced pain models in mice and rats to provide a prospective severity assessment. The WWHow concept integrates intra-operative characteristics for predicting the maximum expected severity of surgical procedures. The assessed severity categorization is mainly congruent with examples in established catalogs; however, because the WWHow concept is based on anatomical location, detailed analysis of the tissue trauma and other intra-operative characteristics, it enables refinement actions, provides the basis for a fact-based dialogue with authority officials and other stakeholders, and helps to identify confounder factors of study findings.

Tmem160 contributes to the establishment of discrete nerve injury-induced pain behaviors in male mice.

Chronic pain is a prevalent medical problem, and its molecular basis remains poorly understood. Here, we demonstrate the significance of the transmembrane protein (Tmem) 160 for nerve injury-induced neuropathic pain. An extensive behavioral assessment suggests a pain modality- and entity-specific phenotype in male Tmem160 global knockout (KO) mice: delayed establishment of tactile hypersensitivity and alterations in self-grooming after nerve injury. In contrast, Tmem160 seems to be dispensable for other nerve injury-induced pain modalities, such as non-evoked and movement-evoked pain, and for other pain entities. Mechanistically, we show that global KO males exhibit dampened neuroimmune signaling and diminished TRPA1-mediated activity in cultured dorsal root ganglia. Neither these changes nor altered pain-related behaviors are observed in global KO female and male peripheral sensory neuron-specific KO mice. Our findings reveal Tmem160 as a sexually dimorphic factor contributing to the establishment, but not maintenance, of discrete nerve injury-induced pain behaviors in male mice.

Combined resting state-fMRI and calcium recordings show stable brain states for task-induced fMRI in mice under combined ISO/MED anesthesia.

For fMRI in animal models, the combination of low-dose anesthetic, isoflurane (ISO), and the sedative medetomidine (MED) has recently become an advocated regimen to achieve stable neuronal states and brain networks in rats that are required for reliable task-induced BOLD fMRI. However, in mice the temporal stability of neuronal states and networks in resting-state (rs)-fMRI experiments during the combined ISO/MED regimen has not been systematically investigated. Using a multimodal approach with optical calcium (Ca2+) recordings and rs-fMRI, we investigated cortical neuronal/astrocytic Ca2+activity states and brain networks at multiple time points while switching from anesthesia with 1% ISO to a combined ISO/MED regimen. We found that cortical activity states reached a steady-state 45 min following start of MED infusion as indicated by stable Ca2+ transients. Similarly, rs-networks were not statistically different between anesthesia with ISO and the combined ISO/MED regimen 45 and 100 min after start of MED. Importantly, during the transition time we identified changed rs-network signatures that likely reflect the different mode of action of the respective anesthetic; these included a dose-dependent increase in cortico-cortical functional connectivity (FC) presumably caused by reduction of ISO concentration and decreased FC in subcortical arousal nuclei due to MED infusion. Furthermore, we report detection of visual stimulation-induced BOLD fMRI during the stable ISO/MED neuronal state 45 min after induction. Based on our findings, we recommend a 45-minute waiting period after switching from ISO anesthesia to the combined ISO/MED regimen before performing rs- or task-induced fMRI experiments.

Systematic review and meta-analysis of cannabinoids, cannabis-based medicines, and endocannabinoid system modulators tested for antinociceptive effects in animal models of injury-related or pathological persistent pain.

ABSTRACT: We report a systematic review and meta-analysis of studies that assessed the antinociceptive efficacy of cannabinoids, cannabis-based medicines, and endocannabinoid system modulators on pain-associated behavioural outcomes in animal models of pathological or injury-related persistent pain. In April 2019, we systematically searched 3 online databases and used crowd science and machine learning to identify studies for inclusion. We calculated a standardised mean difference effect size for each comparison and performed a random-effects meta-analysis. We assessed the impact of study design characteristics and reporting of mitigations to reduce the risk of bias. We meta-analysed 374 studies in which 171 interventions were assessed for antinociceptive efficacy in rodent models of pathological or injury-related pain. Most experiments were conducted in male animals (86%). Antinociceptive efficacy was most frequently measured by attenuation of hypersensitivity to evoked limb withdrawal. Selective cannabinoid type 1, cannabinoid type 2, nonselective cannabinoid receptor agonists (including delta-9-tetrahydrocannabinol) and peroxisome proliferator-activated receptor-alpha agonists (predominantly palmitoylethanolamide) significantly attenuated pain-associated behaviours in a broad range of inflammatory and neuropathic pain models. Fatty acid amide hydrolase inhibitors, monoacylglycerol lipase inhibitors, and cannabidiol significantly attenuated pain-associated behaviours in neuropathic pain models but yielded mixed results in inflammatory pain models. The reporting of criteria to reduce the risk of bias was low; therefore, the studies have an unclear risk of bias. The value of future studies could be enhanced by improving the reporting of methodological criteria, the clinical relevance of the models, and behavioural assessments. Notwithstanding, the evidence supports the hypothesis of cannabinoid-induced analgesia.

A proteome signature for acute incisional pain in dorsal root ganglia of mice.

ABSTRACT: After surgery, acute pain is still managed insufficiently and may lead to short-term and long-term complications including chronic postsurgical pain and an increased prescription of opioids. Thus, identifying new targets specifically implicated in postoperative pain is of utmost importance to develop effective and nonaddictive analgesics. Here, we used an integrated and multimethod workflow to reveal unprecedented insights into proteome dynamics in dorsal root ganglia (DRG) of mice after plantar incision (INC). Based on a detailed characterization of INC-associated pain-related behavior profiles, including a novel paradigm for nonevoked pain, we performed quantitative mass-spectrometry-based proteomics in DRG 1 day after INC. Our data revealed a hitherto unknown INC-regulated protein signature in DRG with changes in distinct proteins and cellular signaling pathways. In particular, we show the differential regulation of 44 protein candidates, many of which are annotated with pathways related to immune and inflammatory responses such as MAPK/extracellular signal-regulated kinases signaling. Subsequent orthogonal assays comprised multiplex Western blotting, bioinformatic protein network analysis, and immunolabeling in independent mouse cohorts to validate (1) the INC-induced regulation of immune/inflammatory pathways and (2) the high priority candidate Annexin A1. Taken together, our results propose novel potential targets in the context of incision and, therefore, represent a highly valuable resource for further mechanistic and translational studies of postoperative pain.

Assessing outcome in postoperative pain trials: are we missing the point? A systematic review of pain-related outcome domains reported in studies early after total knee arthroplasty.

ABSTRACT: The management of acute postoperative pain remains suboptimal. Systematic reviews and Cochrane analysis can assist with collating evidence about treatment efficacy, but the results are limited in part by heterogeneity of endpoints in clinical trials. In addition, the chosen endpoints may not be entirely clinically relevant. To investigate the endpoints assessed in perioperative pain trials, we performed a systematic literature review on outcome domains assessing effectiveness of acute pain interventions in trials after total knee arthroplasty. We followed the Cochrane recommendations for systematic reviews, searching PubMed, Cochrane, and Embase, resulting in the screening of 1590 potentially eligible studies. After final inclusion of 295 studies, we identified 11 outcome domains and 45 subdomains/descriptors with the domain "pain"/"pain intensity" most commonly assessed (98.3%), followed by "analgesic consumption" (88.8%) and "side effects" (75.3%). By contrast, "physical function" (53.5%), "satisfaction" (28.8%), and "psychological function" (11.9%) were given much less consideration. The combinations of outcome domains were inhomogeneous throughout the studies, regardless of the type of pain management investigated. In conclusion, we found that there was high variability in outcome domains and inhomogeneous combinations, as well as inconsistent subdomain descriptions and utilization in trials comparing for effectiveness of pain interventions after total knee arthroplasty. This points towards the need for harmonizing outcome domains, eg, by consenting on a core outcome set of domains which are relevant for both stakeholders and patients. Such a core outcome set should include at least 3 domains from 3 different health core areas such as pain intensity, physical function, and one psychological domain.

Investigating the Role of Ly6G+ Neutrophils in Incisional and Inflammatory Pain by Multidimensional Pain-Related Behavioral Assessments: Bridging the Translational Gap.

In recent years, preclinical pain research has failed to develop genuinely new analgesics for clinical use. This fact is reflected by a high number of patients, limited drug efficacy accompanied by side effects, and a long-term opioid intake. Two main aspects have been addressed, which hinder translation: the use of non-relevant pain models and a mismatch between pain-related outcomes in preclinical and clinical studies. Conversely, disease-specific pain models that mirror more closely the clinical situation and multidimensional behavioral outcome measures that objectively and reproducibly assess relevant pain-related symptoms in a preclinical setting could improve translation. Mechanistically, a matter of debate is the role of Ly6G+ neutrophil granulocytes (NGs) for pain. NGs are essential to eliminate pathogens and promote the wound healing process. For this purpose, there is a need to release various pro- and anti-inflammatory mediators, some of which could ameliorate or enhance pain. However, the contribution of NGs to different pain entities is contradictory for reflex-based tests, and completely unknown in the context of non-evoked pain (NEP) and movement-evoked pain (MEP). First, we combined withdrawal reflex-based assays with novel video-based assessments for NEP- and MEP-related behavior in two mouse pain models. The pain models utilized in this study were incision (INC) and pathogen/adjuvant-induced inflammation (CFA), translating well to postsurgical and inflammatory pain entities. Second, we depleted NGs and applied a set of behavioral assessments to investigate the role of NG migration in different pain modalities. Our comprehensive behavioral approach identified pain-related behaviors in mice that resemble (NEP) or differentiate (MEP) behavioral trajectories in comparison to mechanical and heat hypersensitivity, thereby indicating modality-dependent mechanisms. Further, we show that injury-induced accumulation of NGs minimally affects pain-related behaviors in both pain models. In conclusion, we report a novel assessment to detect NEP in mice after unilateral injuries using a more unbiased approach. Additionally, we are capable of detecting an antalgic gait for both pain entities with unique trajectories. The different trajectories between MEP and other pain modalities suggest that the underlying mechanisms differ. We further conclude that NGs play a subordinate role in pain-related behaviors in incisional and inflammatory pain.

The role of the spinal cyclooxygenase (COX) for incisional pain in rats at different developmental stages.

BACKGROUND: Cyclooxygenase enzymes (COX)-1 and COX-2 are important targets for pain relief after surgery, but the spinal contribution of both isoforms is still unclear, e.g., from a developmental point of view. Here, we studied changes of spinal COX-1 and COX-2 expression and their functional relevance in rats of different ages for pain-related behaviour after incision. METHODS: Mechanical paw withdrawal thresholds (PWT) were assessed before and after incision and after intrathecal administration (IT) of SC-560 (COX-1 inhibitor) or NS-398 (COX-2 inhibitor) in rats aged 5, 14 and 28 days (P5, P14, P28). Furthermore, spinal expressions of COX m-RNA and proteins were investigated. RESULTS: In P5 rats, only IT-administered NS-398 but not SC-560 significantly reversed the decreased PWT after incision. In P14 rats, none of the substance modified PWT, and in P28 rats, only SC-560 increased PWT. Spinal COX-2 mRNA and protein were increased in P5 but not in P14 and P28 rats after incision. Whereas COX-2 is located in spinal neurons, COX-1 is mainly found in spinal microglia cells. CONCLUSION: Our results demonstrate a possible developmental transition from COX-2 to COX-1 activation. Whereas in adult rats spinal COX-1 but not COX-2 is involved in pain-related behaviour after incision, it seems opposite in P5 rats. Interestingly, in P14, neither COX-1 nor COX-2 seems to play a role. This switch may relate to altered neuronal/microglia activation. Our findings indicate specific mechanisms to pain after incision that are age-dependent and may guide further research improving paediatric pain management. SIGNIFICANCE: Postoperative pain in pediatric patients after surgery is still poorly controlled; this might contribute to long-lasting alteration in the nociceptive system and prolonged chronic pain. Here we show a possible developmental switch in the COX-dependent pathway for nociceptive spinal transmission that may explain why pain management in young children needs to be related to age-dependent mechanisms.

Development of a Stimulator for the Characterization of Mechanical-Evoked Pain-Related Supra-Spinal Processing Using BOLD-fMRI in Rodents.

OBJECTIVE: Investigation of pain-related brain processing in animal models is often performed with unspecific stimuli that are not representative of clinically relevant pain phenomena such as punctate hyperalgesia or pressure pain. In order to explore cerebral processing of mechanically evoked pain with functional Magnetic Resonance Imaging (fMRI), a MRI-compatible spatially- and strength-specific mechanical stimulator incorporating either von-Frey filaments or an air-puff system was developed. With this device, mechanical stimuli can be applied to various aspects on the rat hind paw (HP). METHODS: The mechanical stimulator consists of an electro-pneumatic unit connected to the part delivering the mechanical stimulation (MS) via a non-magnetic spring for punctate MS (using a von-Frey filament) or via a Lure-lock unit for pressure pain (air-puffs). The strengths of stimuli were calibrated against the delivered air pressure and weight exerted on a pressure pad or in vivo. BOLD fMRI was performed in a 9.4T MRI scanner during calibrated MS at increasing air pressure. RESULTS: We observed a linear relationship between air pressure and force. These calibrations provided quantitative, adjustable, precise and reproducible sub- and supra-threshold MS. Changes in brain activation were investigated up to a force of 154 g using von-Frey filaments, while the maximum force was 30.9 g for air-puffs. Stimulation demonstrated a significant difference between the two types of MS. Unilateral suprathreshold MS induced strong bilateral brain activation in the areas related to pain processing for both types of MS. However, the patterns of brain activity in subcortical areas evoked by von-Frey MS were different from that evoked by air-puffs. CONCLUSION: The precise delivery of calibrated and reproducible punctate and static MS allows for the specific assessment of clinically relevant supra-spinal correlates of pain-related processes using BOLD fMRI. SIGNIFICANCE: A novel device for clinically relevant MS in BOLD fMRI pain studies in rats, providing results that may be directly translated to human pain research.