accomplished as follows. First, key features are identifi ed that refl ect both the amount of change from baseline, or reactivity ( intensity ), and return to baseline, the regulatio n phase ( recovery ) ( Figure 38.3 ). Quantifi cation is typically represented by a mean of multiple meas- ures of change scores (simple time 1 minus time 2), along with the standard deviation. Th e capacity to upregulate or downregulate the response can be represented by the slope of the change from base- line to maximal stress or stress to recovery (Linden et al., 1997 ), calculated using regression coeffi cients. An area under the curve (AUC) measu re has gained attention as a meaningful cumulative measure of reactivity and recovery, as, for instance, for the cortisol awakening response (Pruessner et al., 2003 ). More organized pat- terns of rhythmic physiological signals, such as HR, signal a greater capacity to res pond to environmental demands. Rather than sig- nalling pain, measures of changes in autono mic and central nerv- ous system activity may refl ect the neural capacity to mount a pain response and an appropriate physiological reaction to a painful stimulus. Nociception (A \200erent inputs) Biomarkers of activity of the nociceptive pathway Perception* (Pain experience) Treatment Biomarkers of Pain or e \200ective treatment in children with limited pain expression beha viors (e.g. non-verbal, autistic etc.) Comparisons of responses between two or more groups require consideration of baseline level s. Th e Law of Initial Values asserts that the size of a psychophysiological response is de pendent on the initial baseline level of the measure (Berntson et al., 1994 ; Wilder, 1962 ). Diff erences between groups in responses to pain- ful events may refl ect diff ering bas elines rather than a variable impact of the noxious event itself. For example, ceiling eff ects have been observed; infants already in an aroused state at the onset of a noxious even t may demonstrate a smaller increase in sympathetic arousal and a smaller decrease in paras ympathetic withdrawal (Grunau et al., 2001 ). Expression* (Pain behavior) In \202uenced by expectation, experience, fear, emotion, attention, belief etc. Figure 38.2 Biomarkers along the pain pathway. Pathway from pain perception to pain express ion in humans and potential for biomarkers of pain at each level. Adapted from Eija Kalso, Biomarkers for pain, PAIN , Volume 107, Issue 3, pp. 199 \200\223201, Copyright 2004. Th is \201 gure has been reproduced with permission of the International Association for the Study of Pain (IASP). Th e \201 gure may not be reproduced for any other purpose wi thout permission. Regulation: Change Score from pain to recovery Intensity: Means SD (How much is enough?) 30 25 Reactivity: Change score from baseline (Law of Initial Value) 20 15 10 5 0 Figure 38.3 Quantifying components of the patterns of response to a painful event. Hypothet ical reactivity patterns comparing responses in Group A vs. Group B. Patterns include measu res of: Intensity (magnitude), Reactivity, Regulation, Direction, and Slope . Image courtes y of Tim Oberlander. Baseline lance recovery \200\2235 Slope (regression coe \203cient): re \202ects tendency to upregulate or downregulate resp onses Direction: is an increase the same as a decrease? SECTION 5 measurement of pain 394 Other limits on biomarkers have been reported. A static or slow responding system or one th at has a limited range of responses is less likely to provide satisfactory biomarkers than a dynamic one. Systems with inherent rhythms, such as the HPA diurnal pattern, may bias aga inst observing an acute response to a painful event if one samples during a morning cortiso l peak period. A predictable response pattern is easier to observe when the signal returns to a baseline compared with a response pattern that remains elevated (continuous response) or has an unpredictable or paroxysmal pat- tern following a stressful event (see Figure 38. 4 ). Th us, consid- eration must be given to a biomarker \200\231s typical response pattern and contextual infl uences to appreciate its behavior in a painful context. function are closely linked to systems that modulate pain reactivity (Randich and Maixner, 1984 ) and activation of vagal aff erents can both facilitate and inhibit nociception (Lovi ck, 1993 ). Developmental maturation of the ANS has to be taken into account when investigating HR and HRV. Typically, mean basilar HR among term-born infants increases to a maximum in the fi rs t 2 months of life and then decreases through infancy (Doussard- Roosevelt et al., 1997 ). In contrast, HRV undergoes opposite changes, and aft er the fi rst 2 months of life is gene rally positively correlated with age (Finley and Nugent, 1995 ; Massin and von Bernuth, 199 7 ). HR typically shows an immediate increase follow- ing the noxious event (Craig et al., 1993 ; Oberlander and Saul, 2002 ) and a decline during recovery. However, in infants a sho rt decrease in HR has been described 3 to 5 sec aft er a vaccine, followed by the character istic increase (Johnston and Strada, 1986 ). Heart rate and heart rate variability Th e ANS plays a key part in the homeostatic response to internal demands and external challenges (stress). Th us, measures of ANS function such as HR or HRV are well suited as biomarkers as these are biological signals that refl ect c omponents of the stress and/or pain response systems. HRV refl ects variation in the time b etween heart beats and is a natural occurring physiological phenomenon. ANS function can be easily studied via non-invasive methods and displays a fast (within seconds) response and recovery pattern that can be quantifi ed to yield measures of magnitude, variability, and d irection of the signal (see Figures 38.3 and 38.4 ). Eff ects are mediated via two branches , parasympathetic and sympathetic, and responses can be quantifi ed to yield discrete conti nuous and bi-di- rectional indexes of relationships between both branches (Berntson et al., 1997 ; Grossman, 1992 ). Further, critical measurement and contextual caveats need to be considered in using HRV mea sures. HRV varies with the length of the HR epoch assessed and proximity to the noxious eve nt. HRV increases as the length of the record increases (McIntosh et al., 1993 ; Oberlander et al., 2000 ; Saul et al., 1988 ). See Voss et al. ( 2009 ) for a review of methodologica l considerations in the use of HRV. Cortisol measures Cortisol is the predominant glucocorticoid in humans and it regu- lates h omeostatic stress responses by mobilizing energy to meet the body \200\231s needs, such as elevating blood glucose levels. In response to a stressor, the HPA axis releases cortisol f rom the adrenal cor- tex, thus off ering a key stress biomarker refl ecting both central an d peripheral reactivity. HR, particularly variations linked to respiratory patterns (vagal tone), provides commonly used autonomic biomarkers (Porges, 1992 ). Systems that control autonomic aspects of cardio vascular Salivary cortisol can be non-invasively obtained and has been more widely studied as a biom arker of aff ective and behavioral responses to stress than pain per se (for review, see Di ckerson Homeostasis: balancing demands Disequilibrium Reestablishing homeostasis Continuous response External demands Figure 38.4 Phases and patterns of biomarker reactivity. Hypothetical characteristic behavi ors of a stress/pain biomarkers extending from an undisturbed baseline condition to acute r esponse phase (reactivity) to recovery (regulation of response) period. Image courtesy of T im Oberlander. Paradoxical response Stressor Regulated response Internal demands Recovery CHAPTER 38 biomarkers of pain: physiological indices of pain reactivity in infants and chil dren 395 and Kemeny, 2004 ; Hellhammer et al., 2009 ). Importantly, corti- sol increases take minute s before they can be observed in saliva or blood, thus cortisol is a rather slow or \200 \230long-acting \200\231 component of stress reactivity. although levels were greater preoperatively compared to non- surgical patients in neonates and school-age children. Th is lack of increase has been interpreted as a protection from s urgical stress and postoperative pain by fentanyl/propofol anesthesia (Mirilas et al., 2010 ). Further, one study found signifi cantly lower BE levels in juvenile patients suff ering from migraines or headaches com- pared to healthy controls (Battistella et al., 1996 ), wh ile another study found signifi cantly increased levels in a group of children suf- fering from a painful atopic dermatitis (pruritus) compared to con- trols or children with healed (non-painful) dermatitis (Georgala et al., 1994 ). Similarly, positive and negative associa tions between pain ratings and BE concentrations in children undergoing pain- ful procedure s have been reported (Bachiocco et al., 1995 ; Katz et al., 1982 ). More studies are needed to better understand the BE response to acute and chronic pain in children. Th e cortisol awakening response has been associated with exper- imental pain ratings in ad ults (e.g. Goodin et al., 2012 ). Pain or stressful events reliably elicit a cortisol incre ase in men and women aged 18 or older; however, in children or adolescents, stressful situa - tions or painful experiences such as inoculations or blood draws do not always lead to si gnifi cant increases in cortisol levels (for review, see Gunnar et al., 2009 ). It has been suggested that pain- or stress- induced HPA axis activation in children may vary with the social environment, such as comforting or distracting parental behavior (McCarthy et al., 2 011 ), demonstrating central regulation of the HPA axis and thus explaining diminished or a bsent increases in cortisol in certain situations (Gunnar et al., 2009 ). Th us, cortisol m ay not be a reliable diagnostic marker for pain experience in a pediatric population. Other biomarkers: skin conductance In response to pain, as well as other sources of stress, skin sym- pathetic nerves release acetylcholine, which acts on muscarine receptors leading to increases in skin conductance. Changes in skin conductance react immediately and are de scribed as continu- ous, objective, and more sensitive and specifi c than other currently a vailable biomarkers for assessing pain (Storm, 2008 ). Th ey have been reported to have a h igh sensitivity to pain (about 90%) and moderately high specifi city (about 60%) in both ch ildren and adults (Hullett et al., 2009 ; Ledowski et al., 2007 ), at least for moderate to severe pain (Breimhorst et al., 2011 ; Loggia et al., 2011b ; Munsters et al., 2012 ; Pere ira-da-Silva et al., 2012 ). Studies have demonstrated associations between self-reported p ain and skin conductance fl uc- tuations in postoperative pediatric patients aged 1 to 16 y ears (Hullett et al., 2009 ), as well as poor correlation of skin conduct- ance with self-r eported pain scores (Choo et al., 2010 ). However, cortisol patterns associated with pain exposure early in life may serve as a bioma rker for predicting subsequent HPA axis stress function. Greater cumulative neonatal pain e xposure in pre- term infants was associated with higher basal cortisol levels during a visu al novelty task at 8 months of age (Grunau et al., 2004 ) and early pain exposure, even whe n controlling for morphine exposure, predicts cortisol levels up to school age in children born preterm (Brummelte et al., 2012a ). Th ese studies suggest that exposure to multiple p ainful events during the preterm period may contrib- ute to \200\230resetting \200\231 sub sequent basal arousal systems, which may regulate the HPA axis system. Th ere is increasing recognition that early experience at critical time periods, perhaps manifesting itself thr ough epigenetic eff ects and brain neuroplasticity, can change neurocognitive development ( Meaney, 2010 ; Neville and Bavelier, 2002 ). Recent studies have raised concern about the reliability (Breimhorst et al., 2011 ) and spe cifi city of electrodermal activity. For instance, central sympathetic inhibitory drugs and sympathetic nerve activation like nausea, vomiting, and anxiety may infl uence the skin co nductance index (Storm, 2008 ). Importantly, abundant tactile stimulation and high-sound le vels in the neonatal units yield stress responses similar to heel stick when monitored usin g plasma catecholamine and skin conductance (Bremner and Fitzgerald, 2008 ; Hellerud and St orm, 2002 ; Lagercrantz et al., 1986 ), question- ing its specifi city. In addition, a rece nt study by Valkenburg et al. ( 2012 ) demonstrated that skin conductance peaks could resul t from changes in vital parameters such as skin temperature and may thus be unrelated to pa in. Skin conductance may provide an additional easy to use and non-invasive tool, but more research is needed to confi rm its specifi city and to determine the clinical applicability of the method. Endogenous opioids as markers of pain Endogenous opioids are an important component of the anti-noci- ceptive system (Millan, 2002 ). Plasma beta endorphin (BE) levels are easy to ob tain and are oft en examined as they relate to acute and chronic pain outcomes (Bruehl et a l., 2012 ). It has been suggested that elevated resting plasma BE levels may be a potential biomar- ker for reduced endogenous opioid analgesic capacity (Bruehl et al., 2012 ). BE pr oduces analgesia by binding to opioid receptors (par- ticularly of the mu subtype) at both pre- and postsynaptic nerve terminals in the peripheral nervous system. Th rough a cascade of interactions, binding of BE results in inhibition of the release of tachykinins, particu larly substance P, a key protein involved in the transmission of pain (Stein, 1995 ). In ad ults, it has been shown that the degree of pain experienced by a surgical patient during an d aft er a procedure correlates with plasma BE levels (Sprouse-Blum et al., 2010 ). Further , both pre- and postoperative plasma BE levels cor- relate positively with postoperative pa in severity for various major surgeries (Matejec et al., 2003 ). Th us, BE levels can be el evated in response to increased pain, probably as a result of increased endog- enous analge sia, but increases have also been found in response to exogenous analgesia (El-Sheik and Bo swell, 2004 ), which makes the interpretation of this signal challenging. In \202 ammatory and genetic markers Infl ammatory markers have been associated with pain. However, most of these cytokines are not specifi c to pain, but are released in response t o infl ammation or tissue damage. Nevertheless, altered cytokine levels have been associate d with chronic pain. For instance, Slade et al. ( 2011 ) identifi ed monocyte chemoattracta nt peptide (MCP)-1, interleukin (IL)-1ra, IL-8, and transforming growth factor (TGF) 1 a s potential diagnostic markers and therapeutic In neonates, infants and children, BE levels did not increase signifi cantly aft er (pain-c ontrolled) surgery (Mirilas et al., 2010 ), SECTION 5 measurement of pain 396 targets for pain in patients with temporomandibular disorder. Animal models suggest that al terations in specifi c cytokine levels and their receptors contribute to the development an d maintenance of chronic pain (e.g. Abbadie et al., 2009 ). Th erefore, these mark- ers may represent novel targets for therapeutic intervention. More research is needed to confi rm this association and its specifi city for pain, especially in children. clinical procedures. Th ese methods have been valuable however in understanding long-term c hanges and neural plasticity in response to acute and chronic pain in the developing brain. As functional imaging off ers a non-invasive \200\230window \200\231 into the developing brain, these applications provide promising tools to assess the nature of the pain experien ce in infants and in children with chronic pain and to provide insights into potential trea tments for pain (Borsook et al., 2011b ; Holsti et al., 2011 ; Ranger et al., 2011 ; Sava e t al., 2009 ). However, not unlike the concern with other biomarkers of pain, the specifi c ity of the marker, i.e. the specifi city of the cognitive activation map in response to pai n is still an issue, as there is again overlap with other physiological systems such as aro usal or stress. Recent research on genetic variability and the association of certain genotypes with altere d drug metabolism underlines the importance of taking genetic variants into account when an alys- ing or interpreting biomarkers. Evidence is evolving demonstrating the increased risk of certain genotypes for developing chronic pain. For instance, Diatchenko and colleagues (2005) demonstrated that variants of the gene encoding catecholamine-O-methyltransferase (C OMT), an enzyme that controls levels of adrenaline and other chemicals and is released in r esponse to stress, infl uenced both human pain perception and sensitivity and the risk for develop- ing a chronic pain condition. Intriguingly, the COMT genotype was a stronger predi ctor of overall pain severity than burn size, burn depth, or time from admission to pain in terview assessment in individuals hospitalized aft er thermal burn injury (Orrey et al., 20 12 ). A case \200\223control study of children suff ering from chronic tension-type headach e (CTTH) investigating the Val158Met poly- morphism of the COMT gene found that children ex pressing the Met/Met genotype showed a longer headache history and lower pressure pain thre sholds compared with those with Met/Val or Val/Val genotype, but no increased predispositio n to suff er from CTTH in children (Fernandez-de-las-Penas et al., 2011 ). Studies have dem onstrated altered ANS activity (e.g. heart rate changes) and cortisol levels in children in response to stressors depending on the COMT or serotonin transporter genotype (Armbruster et al., 2011 ; Mueller et al., 2011 ). Gene \200\223environment interactions have been inve stigated (e.g. Mueller et al., 2011 ), with recent studies in adults suggesting that the Va l158Met polymorphism contributes to interindividual diff erences in neural pain-processing by aff ecting cognitive aspects of pain processing (Loggia et al., 2011a ; Schmahl et al., 2012 ). However, further research with children is needed on this topic. More genetic varia nts have been identifi ed in recent years (e.g. in the gene encoding the P2X7 receptor; Sor ge et al., 2012 ) or the endothelin-receptor A (EDNRA; Kleiber et al., 2007 ), but their po tential as markers of pain and as novel targets for therapeutics, particularly in the pedia tric population, remains unknown. Th ese studies broaden our understanding of individual di ff erences in pain sensitivities and may help to identify children at risk for developing c hronic pain. Biomarkers in special populations Premature care and early infancy Contextual variables inh erent to the painful event have important implications for selection and use of biomarkers (Sellam et al., 2011 ). In a NICU setting, greater exposure to painful procedures, sex, and ventilation have been associated with physiological response to heel sticks (Sellam et al. , 2013 ). Health status of the infant demonstra- bly alters the \200\230performance \200 \231 of biomarkers (Goldstein et al., 1998 ). Similarly, mechanical ventilation, reduced lu ng compliance, neuro- logical compromise from birth asphyxia, intraventricular haemor- rhag e, or periventricular leucomalacia have all been associated with altered HRV and parasympat hetic function (Hanna et al., 2000 ; Prietsch et al., 1994 ; van Ravenswaaij-Arts et al., 1 995 ). Not surprisingly, opioid analgesia may infl uence a biomarker \200\231s response to a painf ul stimulus. In preterm and term infants, opio- ids reduce HR responses to noxious events. In neonates, opioids decrease cardiovascular responses to surgical stress (Anand and Hickey , 1992 ). However, response to opioid analgesics varies with the basal state of the infant. When not in pain, opioids produce tachycardia and increased ventricular contractility by w ay of refl ex sympathetic activation. Th ese adverse side eff ects can be prevented by beta -adrenergic blockade (Franz et al., 1982 ; Vatner et al., 1975 ). In contrast, under condit ions of high initial sympathetic activity such as pain or stress, opioids decrease blood pr essure by a centrally mediated sympatholysis (Lowenstein et al., 1969 ). Th ese diff ering, contrary and context-based opioid-mediated eff ects make it essen- tial to account for the se factors when interpreting an infant \200\231s clinical pain response. Interestingly, eve n simple procedures such as routine tactile nursing procedures can modulate HR and behavior al reac- tivity to pain in very preterm infants if they are preformed shortly before a pain ful procedure, suggesting that a heightened state of arousal aff ected modulation of HR in response to pain (Holsti et al., 2006 ). Further, a recent study by Slater et al. ( 2012 ) showed that preterm infants undergoing a tissue-damaging procedure had higher concentration s of uric acid and malondialdehyde in plasma compared to infants not experiencing any tissu e damage, indicat- ing elevated oxidative stress. Malondialdehyde levels were also sig- nif i cantly correlated with pain scores measured with the Premature Infant Pain Profi le, sugg esting a relationship between procedural pain and oxidative stress in preterm infants. Such an association may prove helpful to determine preterm infants \200\231 pain levels in the future, but more research is needed on this topic. Neuroimaging as a biomarker Neuroimaging applications allow the study of human brain func- tion in vivo . Techniques have been developed which off er diff er- ent temporal, spatial, or spectral resolutions. Th ese methods have identifi ed which brain areas are activated du ring a painful proce- dure in the adult human brain and indicate that touch and pain displa y uniquely diff erent evoked potentials (Apkarian et al., 2011 ). Th e major challenge for using neuroimaging as a marker of pain or pain intensity is that most of the available tech niques, such as electroencephalography (EEG), advanced magnetic imaging, or magnetoencephal ography (MEG), require complex instrumen- tation that is very costly and not easily accessi ble for everyday Among preterm infants, sympathetic control appears to domi- nate, while parasympathetic inf l uences become predominant near term and beyond. Th us, in preterm infants, HRV increases with age CHAPTER 38 biomarkers of pain: physiological indices of pain reactivity in infants and chil dren 397 Conclusion While multiple biomarkers are readily available for use in stud- ies of pain rea ctivity, no single biomarker characterizes all aspects of pediatric pain. Pain system funct ion early in life has complex interrelationships with other developmentally sensitive stres s reac- tivity systems and drawing a single conclusion about \200\230pain biomar- ker resp onses \200\231 based on the observation of function of one system is questionable. Typical childhood contexts vary and infl uence a marker to \200\230behave \200\231 diff erently un der diff erent contextual and illness conditions. but remains lower at term compared with term-born infants, sug- gesting continued maturatio nal delay (Porges, 1992 ). Importantly, small for gestational age infants may have a higher HR and lower HRV when compared with appropriate for gestational age term- born infants (Sp assov et al., 1994 ), suggesting that trends in maturation of cardiac autonomic control may be related to both gestational age and level of neurological maturity. Similarly, the HPA axis is still immature in preterm infants and cortisol levels are oft en inappropriately lo w in infants in the NICU (Fernandez et al., 2008 ; Hanna et al., 1993 ; Kari et al., 1996 ) . Further, it is challenging to obtain blood or suffi cient saliva from these infants to me asure cortisol in a timely pattern. From an evolutionary point of view, behavioral responses such as facial expression and cryi ng are still the best indicators of pain, as mothers did not, and still do not, carry aroun d heart rate monitors or EEG equipment. Modern techniques help us to better understand pain perception and consequences of chronic pain in a population- based model, but are oft en n ot reliable in predicting individual responses or outcomes. In contrast, genetic variations such as the COMT or hepatic metabolic factors may prove to be valuable pre- dictors of pai n sensitivity in adults and hold future promise for individualized anesthesia models and pa in treatments. Conceptual age and early pain experience are also important modifi ers. Preterm infants (<2 8 weeks) have been shown to be more responsive to procedures as they progress towards term (i.e. their HR responses become increasingly more vigorous) (Porter et al., 1999 ). Maximum HR response to a heel lance in infants born at 28 weeks, but studied at 4 weeks of extraut erine life, was higher when compared with infants born at 32 weeks \200\231 gestational age (Johnston et al., 1996 ). Diff erences in HR, HRV, and cortisol at various ages may be due to the early adverse experiences such as repeated pain exposure (Grunau et al., 2005 ) and may refl ect a per- manent reprogramming of the ANS or HPA axis, which needs to be taken i nto consideration when trying to interpret a biomarker \200\231s level or response. It is important to recognize that while pain may be a universal human experience it is by n ature a complex and variable phenome- non (Melzack, 1975 ), both within individuals, over t ime, and across populations. Understanding the pain of another person requires considerable careful judgment, including the need to integrate various sources of information, such as self-report, non-verbal expression and evidence of injury or disease. While all experience has biological substrates, the challenge of identifying and measur- ing biomarkers for such a diverse experience as pain is signifi cant, as many factors, such as the cost, non-invas ive accessibility, depend- ence on environmental factors, the specifi city, sensitivity, an d speed of the response pattern, as well as developmental changes and/or delayed maturation of the involved physiological systems need to be considered. Pain biomarkers in the context of developmental disabilities Children with profound intelle ctual or developmental disabilities are oft en non-verbal or otherwise communicatively impa ired which makes it diffi cult to reliably assess pain status through conven- tional means (i.e. self-report). In these cases, facial action coding has been used to successfully iden tify painful experiences during invasive procedures (Grunau et al., 1990 ; LaChapelle et al ., 1999 ; Nader et al., 2004 ; Oberlander et al., 1999 ). Some studies indicate that prior reports of reduced pain sensitivity in these populations arose from failure to use appropri ate assessment procedures rather than insensitivity in these populations (Tordjman et al., 2009 ). For instance, it was believed that sensory processing is reduced and pain is absent or blunted among individuals with intellectual and related neurodevelopmental disorders (I DDs) and chronic self-in- jurious behavior (SIB), but new data suggests higher (not lower) levels of reactivity to an array of calibrated sensory stimuli for individuals with chronic SIB (Symons, 2011 ). Similarly, Tordjam et al. (2009) reported greater heart rate response to venepuncture and the elevated plasma beta-endorphin levels in individuals with autism, which refl ects enhanced physiological and biological stress responses. Th is is consistent with the fi ndings of Nader et al. ( 2004 ) demonstrating more vigorous facial displays du ring venepuncture in children with autism. Th ese results indicate that children with autis m are sensitive to pain, despite prior reports of reduced pain sensitivity (Tordjman et al. , 2009 ). Th ese studies underline the need for reliable physiological biomarkers of pain, which can be used in addition to conventional measures and behavioral observations. Childre n with cognitive or emotional impairments may have dif- ferent modes of expressing their pa in, and having physiological indices of nociception or pain perception available would be a tre- mendous asset to help reduce the suff ering of these patients. Taken together, we are in urgent need of \200\230better \200\231 physiological biomarkers of pain to reliable assess pain in non-verbal or other- wise challenged children that would work towards identifying the best multidimensional approach to understand and eff ectively treat pain in the pediatric patients. Case example: irritability of unknown origin \200\224making sense of biomarker pain signals ? George is a 12-year-old with multiple developmental disabili- ties associated with a signif i cant neurological impairment of an unknown genetic-metabolic disorder. He has multiple co g- nitive, language, and motor impairments, is fed via a gastros- tomy tube, has infrequent seizures, and has been treated for gastro-oesophageal refl ux. He has a ventriculoperitone al shunt for hydrocephalus and is thought to have recurrent headaches \200\224 there is a f amily history of migraines. George \200\231s medications include valproic acid, ranitidine, and lactulose, with glyceri n suppositories, phosphate enemas, acetaminophen, and lorazepam, as needed. His parents and car- egivers rely on a variety of non-specifi c verbal and hand and head movements to unde rstand his communication of pleas- ure or discomfort. Increasingly, he engages in SIB. His symp- toms have increased in frequency, duration, and intensity and SECTION 5 measurement of pain 398 responses reliably discriminate between diff erent stimulus intensities in experimentally i nduced pain ? J Pain , 12 , 61 \200\22370. particularly troubling are his continuous facial grimacing. In the rare moments when he is not crying his parents report that he becomes irritable and typical sensory experiences tha t had been pleasant were now ineff ective or even aversive. One night, George awoke crying with increased vigor. Finding that he had no fever, but his HR was 150/min at rest and he w as breathing rapidly (>40/min), his father stopped an overnight feed. Because of increased generalized arousal and irritability, which his mother thinks refl ects a severe painful co ndition, he was brought to his local emergency department. Bremner , L. R. , and Fitzgerald , M. ( 2008 ). Postnatal tuning of cutaneous inhibitory receptive fi elds in the rat . J Physiol , 586 , 1529 \200\2231537. Bruehl , S. , Burns , J. W. , Chung , O. Y. , and Chont , M. ( 2012 ). What do plasma beta- endorphin levels reveal about endogenous opioid analgesic function? Eur J Pain , 16 , 370 \200\223380. Brummelte , S. , Chau , C. M. , Synnes , A. , Cepeda , I. L. , Weinberg , J. , and Grunau , R. E. ( 2012 a). Neonatal pain predicts cortisol levels at age 7 years in children born pr eterm. Pediatric Academic Societies Meeting, Boston, MA, Abstract 3640.2. Brummelte , S. , Grunau , R. E. , Chau , V. , Poskitt , K. J. , Brant , R. , Vinall , J. , et al . ( 2012 b). Procedural pain and brain development in premature newborns . Ann Neurol , 71 , 385 \200\223396. On examination he was agitated and engaged in SIB (hitting his left ear) coupled with a gri mace. He was afebrile but respon- sive to touch and his heart rate was 80/min and regular, and respiratory rate was 15/min. Laboratory investigations revealed a white blood count of 18 000, with a left shift and erythrocyte sedimentation rate of 45. Chest X-ray was typical and consist- ent with previous images. Th e residents who examined him were puzzled \200 \224he appeared to all as if he might be in pain, but his cardiorespiratory status was tell ing a diff erent story \200\224where were the \200\230pain signals \200\231, they asked? Choo , E. K. , Magruder , W. , Montgomery , C. J. , Lim , J. , Brant , R. , and Ansermino , J. M. ( 2010 ). Skin conductance fl uctuations correlate poorly with postoperative self-re port pain measures in school-aged children . Anesthesiology , 113 , 175 \200\223182. Craig , K. D. , Versloot , J. , Goubert , L. , Vervoort , T. , and Crombez , G. ( 2010 ). P erceiving pain in others: automatic and controlled mechanisms . J Pain , 11 , 101 \200\223 108. Craig , K. D. , Whitfi eld , M. F. , Grunau , R. V. , Linton , J. , and Hadjistavropoulos , H. D. ( 1993 ). 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