Genome-wide association studies with experimental validation identify a protective role for B lymphocytes against chronic post-surgical pain.

BACKGROUND: Chronic post-surgical pain (CPSP) significantly impacts patients' recovery and quality of life. Although environmental risk factors are well-established, genetic risk remains less understood. METHODS: A meta-analysis of genome-wide association studies followed by partitioned heritability was performed on 1350 individuals across five surgery types: hysterectomy, mastectomy, abdominal, hernia, and knee. In subsequent animal studies, withdrawal thresholds to evoked mechanical stimulation were measured in Rag1 null mutant and wild-type mice after plantar incision and laparotomy. Cell sorting by flow cytometry tracked recruitment of immune cell types. RESULTS: We discovered 77 genome-wide significant single-nucleotide polymorphism (SNP) hits, distributed among 24 loci and 244 genes. Meta-analysis of all cohorts estimated a SNP-based narrow-sense heritability for CPSP at ∼39%, indicating a substantial genetic contribution. Partitioned heritability analysis across a wide variety of tissues revealed enrichment of heritability in immune system-related genes, particularly those associated with B and T cells. Rag1 null mutant mice lacking both T and B cells exhibited exacerbated and prolonged allodynia up to 42 days after surgery, which was rescued by B-cell transfer. Recruitment patterns of B cells but not T cells differed significantly during the first 7 days after injury in the footpad, lymph nodes, and dorsal root ganglia. CONCLUSIONS: These findings suggest a key protective role for the adaptive immune system in the development of chronic post-surgical pain.

Maternal and iatrogenic neonatal opioid withdrawal syndrome: Differences and similarities in recognition, management, and consequences.

Opioids are potent analgesics used to manage pain in both young and old, but the increased use in the pregnant population has significant individual and societal implications. Infants dependent on opioids, either through maternal or iatrogenic exposure, undergo neonatal opioid withdrawal syndrome (NOWS), where they may experience withdrawal symptoms ranging from mild to severe. We present a detailed and original review of NOWS caused by maternal opioid exposure (mNOWS) and iatrogenic opioid intake (iNOWS). While these two entities have been assessed entirely separately, recognition and treatment of the clinical manifestations of NOWS overlap. Neonatal risk factors such as age, genetic predisposition, drug type, and clinical factors like type of opioid, cumulative dose of opioid exposure, and disease status affect the incidence of both mNOWS and iNOWS, as well as their severity. Recognition of withdrawal is dependent on clinical assessment of symptoms, and the use of clinical assessment tools designed to determine the need for pharmacotherapy. Treatment of NOWS relies on a combination of non-pharmacological therapies and pharmacological options. Long-term consequences of opioids and NOWS continue to generate controversy, with some evidence of anatomic brain changes, but conflicting animal and human clinical evidence of significant cognitive or behavioral impacts on school-age children. We highlight the current knowledge on clinically relevant recognition, treatment, and consequences of NOWS, and identify new advances in clinical management of the neonate. This review brings a unique clinical perspective and critically analyzes gaps between the clinical problem and our preclinical understanding of NOWS.

The development of descending serotonergic modulation of the spinal nociceptive network: a life span perspective.

The nociceptive network, responsible for transmission of nociceptive signals that generate the pain experience, is not fully developed at birth. Descending serotonergic modulation of spinal nociception, an important part of the pain network, undergoes substantial postnatal maturation and is suggested to be involved in the altered pain response observed in human newborns. This review summarizes preclinical data of the development of descending serotonergic modulation of the spinal nociceptive network across the life span, providing a comprehensive background to understand human newborn pain experience and treatment. Sprouting of descending serotonergic axons, originating from the rostroventral medulla, as well as changes in receptor function and expression take place in the first postnatal weeks of rodents, corresponding to human neonates in early infancy. Descending serotonergic modulation switches from facilitation in early life to bimodal control in adulthood, masking an already functional 5-HT inhibitory system at early ages. Specifically the 5-HT3 and 5-HT7 receptors seem distinctly important for pain facilitation at neonatal and early infancy, while the 5-HT1a, 5-HT1b, and 5-HT2 receptors mediate inhibitory effects at all ages. Analgesic therapy that considers the neurodevelopmental phase is likely to result in a more targeted treatment of neonatal pain and may improve both short- and long-term effects. IMPACT: The descending serotonergic system undergoes anatomical changes from birth to early infancy, as its sprouts and descending projections increase and the dorsal horn innervation pattern changes. Descending serotonergic modulation from the rostral ventral medulla switches from facilitation in early life via the 5-HT3 and 5-HT7 receptors to bimodal control in adulthood. A functional inhibitory serotonergic system mainly via 5-HT1a, 5-HT1b, and 5-HT2a receptors at the spinal level exists already at the neonatal phase but is masked by descending facilitation.

Identifying the Neurodevelopmental Differences of Opioid Withdrawal.

Stopping opioid medications can result in a debilitating withdrawal syndrome in chronic users. Opioid withdrawal can occur at all ages, but mechanistic understanding of this condition is predominantly derived from adult studies. Here, we examined whether there are age-dependent differences in the behavioural phenotype and cellular indices of opioid withdrawal. We tested this by assessing the behavioural and cFos response (a surrogate marker for neuronal activation) to morphine withdrawal in C57BL/6J mice across key developmental stages-neonatal, adolescent, and adulthood. Mice in all age groups received escalating doses of morphine (10-50 mg/kg) over 5 days and withdrawal was precipitated by a single injection of the opioid receptor antagonist naloxone (2 mg/kg) two hours after the last morphine dose. In adult and adolescent mice, withdrawal behaviours were robust, with age-related differences in autonomic and somatic signs. In both groups, cFos expression was increased in spinally projecting neurons within the Periaqueductal Grey (PAG), Rostro-ventromedial Medulla (RVM), and Locus Coeruleus. Neonatal animals displayed both a distinct behavioural withdrawal and cFos expression profile. Notably, in young animals cFos expression was increased within the PAG and LC, but decreased in the RVM. In summary, naloxone challenge precipitated robust opioid withdrawal behaviours across all developmental stages with neonatal animals displaying differences in withdrawal behaviours and unique neuronal activation patterns within key brainstem regions.

Methadone effectively attenuates acute and long-term consequences of neonatal repetitive procedural pain in a rat model.

BACKGROUND: Painful procedures in early life cause acute pain and can alter pain processing at a spinal level lasting into adulthood. Current methods of analgesia seem unable to prevent both acute and long-term hypersensitivity associated with neonatal pain. The current study aims to prevent acute and long-term hypersensitivity associated with neonatal procedural pain using methadone analgesia in rat pups. METHODS: Sprague-Dawley rat pups received either methadone (1 mg/kg) or saline prior to repetitive needle pricks into the left hind paw from the day of birth (postnatal day (P)0) to P7. Control littermates received a tactile stimulus. Mechanical sensitivity was assessed during the neonatal period (P0-P7), from weaning to adulthood (3-7 weeks) and following surgical re-injury of the same dermatome in adulthood. RESULTS: Methadone administration completely reversed acute hypersensitivity from P0 to P7. In addition, neonatal methadone analgesia prevented prolonged hypersensitivity after re-injury in adulthood, without affecting sensitivity from weaning to adulthood. CONCLUSIONS: The current study shows that neonatal methadone analgesia can attenuate acute as well as long-term hypersensitivity associated with neonatal procedural pain in a rat model. IMPACT: Methadone treatment attenuates acute and long-term hypersensitivity associated with neonatal pain in a rat model. Clinical effectiveness studies are urgently warranted to assess acute and long-term analgesic effectivity of methadone.

Neonatal procedural pain affects state, but not trait anxiety behavior in adult rats.

The influence of neonatal experiences upon later-life affective behavior is increasingly recognized, but the reported effects on anxiety are often contradictory. The observed effect may depend upon the type of anxiety (state or trait) affected. The current study aims to investigate whether neonatal repetitive needle pricking alters anxiety behavior in adulthood, by assessing both state and trait anxiety in rats. Sprague-Dawley rat pups received four unilateral needle pricks per day, while controls received four tactile stimuli or were left completely undisturbed during the first postnatal week. Mechanical sensitivity was assessed in the neonatal phase and throughout the development. State anxiety was assessed in the open field test and trait anxiety in the elevated zero maze. The results show that repetitive needle pricking leads to acute mechanical hypersensitivity, but does not affect baseline mechanical sensitivity throughout development. In adulthood, animals previously exposed to neonatal procedural pain (including repetitive handling and removal from litter) showed lower state anxiety but did not differ in trait anxiety, as compared with the undisturbed controls. These findings indicate that early-life procedural pain decreases state but not trait anxiety behavior in later life in a rodent model of repetitive needle pricking.

Polygenic risk scores indicates genetic overlap between peripheral pain syndromes and chronic postsurgical pain.

Chronic postsurgical pain (CPSP) is a debilitating chronic pain condition that has a substantial effect on quality of life. CPSP shows considerable clinical overlap with different chronic peripheral pain syndromes, suggesting a shared aetiology. This study aims to assess the genetic overlap between different chronic pain syndromes and CPSP, providing relevant biological context for potential chronic pain markers of CPSP. To analyse the genetic overlap between CPSP and chronic peripheral pain syndromes, recent GWAS studies were combined for polygenic risk scores (PRS) analysis, using a cohort of CPSP patients as starting point. Biological contextualisation of genetic marker, overlap between CPSP and chronic pain syndromes, was assessed through Gene Ontology (GO), using Pathway Scoring Algorithm (PASCAL) and REVIGO. PRS analyses suggest a significant genetic overlap between CPSP and 3 chronic pain disorders: chronic widespread pain (CWP, p value threshold = 0.003, R2 0.06, p = 0.003), rheumatoid arthritis (RA, p value threshold = 0.0177, R2 = 0.04, p = 0.017) and possibly sciatica (p value threshold = 0.00025, R2 = 0.03, p = 0.045). Whereas no significant genetic overlap was found with cluster headache and migraine, the outcome for osteoarthritis (OA) was inconsistent between the cohorts. This is likely related to cohort composition, as repeated random reallocation of patients' nullified CPSP/OA outcome variation between the discovery and replication cohorts. GO analyses suggested an aetiological involvement of genetic markers that control neurological signalling (specifically sodium channels) and inflammatory response. The current study reaffirms the impact of sample size, cohort composition and open data accessibility on the unbiased identification of genetic overlap across disorders. In conclusion, this study is the first to report genetic overlap between regulatory processes implicated in CPSP and chronic peripheral pain syndromes. Interaction between neurological signalling and inflammatory response may explain the genetic overlap between CPSP, CWP and RA. Enhanced understanding of mechanisms underlying chronification of pain will aid the development of new therapeutic strategies for CPSP with sodium channel biochemistry as a potential candidate.

Dopaminergic neurotransmission and genetic variation in chronification of post-surgical pain.

Chronic post-surgical pain (CPSP) is a debilitating condition affecting 10-50% of surgical patients. The current treatment strategy for CPSP is not optimal, and the identification of genetic variation in surgical patients might help to improve prediction and treatment of CPSP. The neurotransmitter dopamine (DA) has been associated with several chronic pain disorders. This narrative review focuses on DA neurotransmission as a potential target in the treatment of CPSP. The current knowledge on genetic variation within DA neurotransmission and its role in CPSP susceptibility are reviewed. Three genes involved in DA neurotransmission (COMT, GCH1, and DRD2) have been associated with variability in pain sensitivity, development of CPSP, and analgesic requirement. The direction of the effect of the association is sometimes inconclusive because of contradictory results, but ample evidence suggests a modulatory role of DA. Because of this modulatory role, DA is an excellent pharmacological target in the treatment of pain. Pharmacotherapy focused on DA neurotransmission has potential in both prevention (via D1-like receptors) and treatment (via D2-like receptors and DA reuptake inhibitors) of CPSP. The development of prediction models including genetic risk factors is necessary to better identify patients at risk.

Genetic polymorphisms and prediction of chronic post-surgical pain after hysterectomy-a subgroup analysis of a multicenter cohort study.

BACKGROUND: Chronic post-surgical pain (CPSP) is a serious problem. Clinical and psychological variables have not been able to explain all observed variance in prevalence and severity of CPSP. The first objective is to determine the association between genetic polymorphisms and the prevalence of CPSP after hysterectomy. The second objective is to analyze if the implementation of genetic polymorphisms into a previously performed clinical and psychological predictor analysis on the development of CPSP after hysterectomy will improve its discriminatory power. METHODS: A prospective multicenter cohort study was performed in patients undergoing hysterectomy for benign indication. Clinical and psychological variables were collected by questionnaires in the week before surgery, post-operatively up to day 4, 3 and 12 months after hysterectomy. Blood was collected and 16 polymorphisms previously suggested to be correlated to CPSP (COMT, GCH1, KCNS1, CACNG2, and OPRM1) were genotyped. Logistic regression analyses were performed. RESULTS: Three hundred and forty-five patients were available for the genetic analyses. The prevalence of CPSP 3 months post-operatively was 10.5% and after 12 months 7.9%. The polymorphism rs4818 within the COMT gene was associated with the prevalence of CPSP after 3 months. No polymorphisms were associated with CPSP after 12 months. The addition of rs4818 to the prediction model did not change its discriminatory power significantly. CONCLUSION: The rs4818 polymorphism within the COMT gene was associated with the prevalence of CPSP 3 months after hysterectomy, but the implementation of rs4818 into the prediction model did not significantly improve the chance of identifying hysterectomy patients at risk for CPSP.

Targeting Pannexin-1 Channels: Addressing the 'Gap' in Chronic Pain.

Chronic pain complicates many diseases and is notoriously difficult to treat. In search of new therapeutic targets, pannexin-1 (Panx1) channels have sparked intense interest as a key mechanism involved in a variety of chronic pain conditions. Panx1 channels are transmembrane proteins that release ions and small molecules, such as adenosine triphosphate (ATP). They are expressed along important nodes of the pain pathway, modulating activity of diverse cell types implicated in the development and progression of chronic pain caused by injury or pathology. This review highlights advances that have unlocked the core structure and machinery controlling Panx1 function with a focus on understanding and treating chronic pain.

Genome-wide association study suggests a critical contribution of the adaptive immune system to chronic post-surgical pain.

Chronic post-surgical pain affects a large proportion of people undergoing surgery, delaying recovery time and worsening quality of life. Although many environmental variables have been established as risk factors, less is known about genetic risk. To uncover genetic risk factors we performed genome-wide association studies in post-surgical cohorts of five surgery types- hysterectomy, mastectomy, abdominal, hernia, and knee- totaling 1350 individuals. Genetic associations between post-surgical chronic pain levels on a numeric rating scale (NRS) and additive genetic effects at common SNPs were evaluated. We observed genome-wide significant hits in almost all cohorts that displayed significance at the SNP, gene, and pathway levels. The cohorts were then combined via a GWAS meta-analysis framework for further analyses. Using partitioned heritability, we found that loci at genes specifically expressed in the immune system carried enriched heritability, especially genes related to B and T cells. The relevance of B cells in particular was then demonstrated in mouse postoperative pain assays. Taken altogether, our results suggest a role for the adaptive immune system in chronic post-surgical pain.

Selective Targeting of Serotonin 5-HT1a and 5-HT3 Receptors Attenuates Acute and Long-Term Hypersensitivity Associated With Neonatal Procedural Pain.

Neonatal painful procedures causes acute pain and trigger long-term changes in nociceptive processing and anxiety behavior, highlighting the need for adequate analgesia during this critical time. Spinal serotonergic receptors 5-HT1a and 5-HT3 play an important role in modulating incoming nociceptive signals in neonates. The current study aims to attenuate acute and long-term hypersensitivity associated with neonatal procedural pain using ondansetron (a 5-HT3 antagonist) and buspirone (a 5-HT1a agonist) in a well-established rat model of repetitive needle pricking. Sprague-Dawley rat pups of both sexes received ondansetron (3 mg/kg), buspirone (3 mg/kg) or saline prior to repetitive needle pricks into the left hind-paw from postnatal day 0-7. Control animals received tactile stimulation or were left undisturbed. Acute, long-term, and post-operative mechanical sensitivity as well as adult anxiety were assessed. Neonatal 5-HT1a receptor agonism completely reverses acute hypersensitivity from P0-7. The increased duration of postoperative hypersensitivity after re-injury in adulthood is abolished by 5-HT3 receptor antagonism during neonatal repetitive needle pricking, without affecting baseline sensitivity. Moreover, 5-HT1a and 5-HT3 receptor modulation decreases adult state anxiety. Altogether, our data suggests that targeted pharmacological treatment based on the modulation of spinal serotonergic network via the 5-HT1a and 5-HT3 receptors in neonates may be of use in treatment of neonatal procedural pain and its long-term consequences. This may result in a new mechanism-based therapeutic venue in treatment of procedural pain in human neonates.

Anatomical changes in descending serotonergic projections from the rostral ventromedial medulla to the spinal dorsal horn following repetitive neonatal painful procedures.

Excessive noxious stimulation during the critical neonatal period impacts the nociceptive network lasting into adulthood. As descending serotonergic projections from the rostral ventromedial medulla (RVM) to the spinal dorsal horn develop postnatally, this study aims to investigate the long-term effect of repetitive neonatal procedural pain on the descending serotonergic RVM-spinal dorsal horn network. A well-established rat model of repetitive noxious procedures is used in which neonatal rats received four noxious needle pricks or tactile stimulation with a cotton swab per day in the left hind paw from day of birth to postnatal Day 7. Control animals were left undisturbed. When animals reached adulthood, tissue was collected for quantitative immunohistochemical analysis of serotonin (5-hydroxytryptamine, 5-HT) in the RVM and spinal dorsal horn. Both repetitive noxious and tactile procedures in the neonate decreased the 5-HT staining intensity in the adult ipsilateral but not contralateral spinal dorsal horn. Repetitive neonatal noxious procedures resulted in an increased area covered with 5-HT staining in the adult RVM ipsilateral to the side of injury, whereas repetitive neonatal tactile stimulation resulted in increased 5-HT staining intensity in both the ipsi- and contralateral RVM. The number of 5-HT cells in adult RVM is unaffected by neonatal conditions. This detailed anatomical study shows that not only neonatal noxious procedures but also repetitive tactile procedures result in long-lasting anatomical changes of the descending serotonergic system within the RVM and spinal dorsal horn. Future studies should investigate whether these anatomical changes translate to functional differences in descending serotonergic modulation after neonatal adverse experiences.

Cannabinoids in Chronic Pain: Therapeutic Potential Through Microglia Modulation.

Chronic pain is a complex sensory, cognitive, and emotional experience that imposes a great personal, psychological, and socioeconomic burden on patients. An estimated 1.5 billion people worldwide are afflicted with chronic pain, which is often difficult to treat and may be resistant to the potent pain-relieving effects of opioid analgesics. Attention has therefore focused on advancing new pain therapies directed at the cannabinoid system because of its key role in pain modulation. Endocannabinoids and exogenous cannabinoids exert their actions primarily through Gi/o-protein coupled cannabinoid CB1 and CB2 receptors expressed throughout the nervous system. CB1 receptors are found at key nodes along the pain pathway and their activity gates both the sensory and affective components of pain. CB2 receptors are typically expressed at low levels on microglia, astrocytes, and peripheral immune cells. In chronic pain states, there is a marked increase in CB2 expression which modulates the activity of these central and peripheral immune cells with important consequences for the surrounding pain circuitry. Growing evidence indicate that interventions targeting CB1 or CB2 receptors improve pain outcomes in a variety of preclinical pain models. In this mini-review, we will highlight recent advances in understanding how cannabinoids modulate microglia function and its implications for cannabinoid-mediated analgesia, focusing on microglia-neuron interactions within the spinal nociceptive circuitry.

Dopamine-neurotransmission and nociception in zebrafish: An anti-nociceptive role of dopamine receptor drd2a.

Dopamine (DA) is an important modulator in nociception and analgesia. Spinal DA receptors are involved in descending modulation of the nociceptive transmission. Genetic variations within DA neurotransmission have been associated with altered pain sensitivity and development of chronic pain syndromes. The variant rs6277 in dopamine receptor 2 a (drd2a) has been associated with a decreased D2 receptor availability and increased nociception. The aim of this study is to further characterize the role of DA neurotransmission in nociception and the anti-nociceptive function of drd2a. The phenotype caused by rs6277 was modelled in zebrafish larvae using morpholino's and the effect on nociception was tested using a validated behavioural assay. The anti-nociceptive role of drd2a was tested using pharmacological intervention of D2 agonist Quinpirole. The experiments demonstrate that a decrease in drd2a expression results in a pro-nociceptive behavioural phenotype (P = 0.016) after a heat stimulus. Furthermore, agonism of drd2a with agonist Quinpirole (0.2 µM) results in dose-dependent anti-nociception (P = 0.035) after a heat stimulus. From these results it is concluded that the dopamine receptor drd2a is involved in anti-nociceptive behaviour in zebrafish. The model allows further screening and testing of genetic variation and treatment involved in nociception.

Developmental neurobiology as a guide for pharmacological management of pain in neonates.

Pain in newborn children should be prevented due to negative short- and long-term consequences. A good understanding of the development of the nociceptive system in newborns is necessary to enable optimal pain assessment, and most importantly to treat and prevent pain adequately in neonates. So far, preclinical juvenile animal studies have led to a tremendous amount of information regarding the development of the nociceptive system. In addition, they have made clear that the developmental stage of the nociceptive system may influence the mechanism of action of different classes of analgesics. Age specific analgesic therapy, based on post-menstrual age, should therefore be considered by incorporating information on the developmental stages of the nociceptive system in combination with knowledge from pharmacokinetic and -dynamic studies in neonates.

Repeated touch and needle-prick stimulation in the neonatal period increases the baseline mechanical sensitivity and postinjury hypersensitivity of adult spinal sensory neurons.

Noxious stimulation at critical stages of development has long-term consequences on somatosensory processing in later life, but it is not known whether this developmental plasticity is restricted to nociceptive pathways. Here, we investigate the effect of repeated neonatal noxious or innocuous hind paw stimulation on adult spinal dorsal horn cutaneous mechanical sensitivity. Neonatal Sprague-Dawley rats of both sexes received 4 unilateral left hind paw needle pricks (NPs, n = 13) or 4 tactile (cotton swab touch) stimuli, per day (TC, n = 11) for the first 7 days of life. Control pups were left undisturbed (n = 17). When adult (6-8 weeks), lumbar wide-dynamic-range neuron activity in laminae III-V was recorded using in vivo extracellular single-unit electrophysiology. Spike activity evoked by cutaneous dynamic tactile (brush), pinch and punctate (von Frey hair) stimulation, and plantar receptive field areas were recorded, at baseline and 2 and 5 days after left plantar hind paw incision. Baseline brush receptive fields, von Frey hair, and pinch sensitivity were significantly enhanced in adult NP and TC animals compared with undisturbed controls, although effects were greatest in NP rats. After incision, injury sensitivity of adult wide-dynamic-range neurons to both noxious and dynamic tactile hypersensitivity was significantly greater in NP animals compared with TC and undisturbed controls. We conclude that both repeated touch and needle-prick stimulation in the neonatal period can alter adult spinal sensory neuron sensitivity to both innocuous and noxious mechanical stimulation. Thus, spinal sensory circuits underlying touch and pain processing are shaped by a range of early-life somatosensory experiences.

Adult spinal opioid receptor µ1 expression after incision is altered by early life repetitive tactile and noxious procedures in rats.

Clinical and experimental data suggests that noxious stimulation at critical stages of development results in long-term changes on nociceptive processing in later life. Here, we use an established, well-documented rat model of repetitive noxious procedures closely mimicking the clinical situation in the NICU. In order to understand molecular changes underlying the long-term consequences of repetitive stimulation of the developing nociceptive system the present study aims to analyze the presence of the µ-opioid-receptor-1 (OPRM1). Neonatal rats received either four needle pricks per day in the left hind-paw from postnatal day 0-7 as a model of procedural pain in infancy. Control pups were handled in the same way but were instead tactile stimulated, or were left undisturbed. At the age of 8 weeks, all animals received an ipsilateral hind-paw incision as a model for post-operative pain, and mechanical sensitivity was tested at multiple time-points. Before, and 1 or 5 days post-incision, spinal cord tissue was collected for immunostaining of opioid receptor OPRM1. Semi-quantitative immunocytochemical analysis of superficial laminae in lumbar spinal dorsal horn revealed that: (1) early life repetitive tactile or noxious procedures do not alter baseline levels of OPRM1 staining intensity and (2) early life repetitive tactile or noxious procedures lead to a decrease in OPRM1 staining intensity 5 days after incision in adulthood compared to undisturbed controls. We conclude that early life repetitive tactile or noxious procedures affect the intensity of OPRM1-immunoreactivity in the lumbar superficial spinal cord dorsal horn after adulthood injury, without affecting baseline intensity. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 78: 417-426, 2018.