Electroencephalography and evoked potentials Electroencephalography (EEG) has been extensiv ely used to inves- tigate neurological function in neonates and infants. Th e technique pro vides an opportunity to record the electrical activity generated by large populations of ce rebral neurons. An EEG represents fl uctu- ating electrical fi eld potentials as a function of time, and the defl ec- tions represent changes in voltage and polarity. By recording fr om multiple electrode sites, it is possible to obtain a representation of the spatial distr ibution of the electric fi eld recorded over the scalp. EEG is oft en characterized by the frequency and amplitude of the recorded activity and during the early neonatal period the c harac- teristic patterns of EEG activity change rapidly with gestational age (Andr et al ., 2010 ). For example, throughout early developmen- tal there is a reduction in the discon tinuity observed in the EEG recordings. In addition, an increase in characterized rhythmica l patterns of activity, such as delta brushes and temporal sawtooth waveforms, is observed, followed by their subsequent disappear- ance (Dreyfus-Brisac and Monod, 1972 ). Th e ident ifi cation of these characteristic patterns of EEG activity during this developmental perio d is oft en used in the clinical assessment of neonatal cerebral function. NIRS is widely used in neonatal research to measure functional activation of the cortex (Ku saka et al., 2004 ). Recently, this tech- nique has been used in neonatal studies investiga ting cerebral responses following noxious stimulation. Cortical haemodynamic activity has b een recorded in infants in response to noxious events, including heel lance, venepuncture, and endotracheal tube suc- tioning and repositioning (Bartocci et al., 2006 ; Limperopoulos et al., 2008 ; Slater et al., 2006 , 2008 ). Th ese studies suggest that an increase in he moglobin concentration can be observed in the CHAPTER 39 the neurophysiological evaluation of nociceptive responses in neonates 403 EEG can be used to measure specifi c patterns of brain activity recorded in response to kno wn stimuli. Th ese evoked patterns of activity are referred to as evoked or event-related p otentials. In neonatal pain research, specifi c changes in EEG activity have been recorded in response to acute noxious stimulation, such as a clini- cally essential heel lance (Slat er et al., 2010a , 2010b ). By considering the features of the evoked activity, such as the polarity, timing, and topography, inference can be made about the how the brain proc- esse s noxious sensory information. telencephalon, which subsequently develops into the cerebral cor- tex and basal ganglia (Ko stovic and Rakic, 1990 ; see also Fitzgerald, Chapter 8, this volume). From the subplate gr ows the thalamo-cor- tical and cortico-cortical connections (Kostovic and Rakic, 1990 ), wi th its neurons contributing to the fi rst evoked potentials from 24 weeks of gestation (Hrb ek et al., 1973 ). Standard MRI techniques allow for the assessment of brain development in infants. Highly sensitive assessment of gray and white matter and the diff erentia- tion o f myelination in white matter can be visualized. In vivo , the transient subplate zone can be seen as an area of hypointensity on magnetic resonance images (Prayer et al., 2006 ), bu t aft er 32 weeks of gestation, the transient subplate zone becomes less visible (Kostovic and Vasung, 2009 ). An event-locking technique has been developed to enable the evoked neuronal activity follow ing a single clinical noxious event to be recorded (Fabrizi et al., 2011 ; Worley et al., 2 012 ). Use of this technique has provided electrophysiological evidence that the immature i nfant brain can discriminate between acute noxious and non-noxious stimulation from 35 gest ational weeks (Slater et al., 2010a ). Th ese electrophysiological measures of nociceptive- spe- cifi c brain activity are useful for understanding how the developing human brain resp onds to nociceptive stimuli, and may potentially be useful to test the ability of analgesic treatments in neonates to suppress these increases in brain activity. Nevertheless, these exper- imental studies are challenging and require specialist data acquisi- tion and analys is skills (Fabrizi et al., 2011 ; Vanhatalo et al., 2009 ; Worley et al., 2012 ). Functional MRI (fMRI) is a non-invasive technique that assesses cortical activation. Th e m ost commonly used contrast method is blood oxygen level-dependent (BOLD) imaging. BOLD meas ures the magnetic change in hemoglobin between the oxygenated and deoxygenated states that accompany neuronal activity (Logothetis and Pfeuff er, 2004 ; Ogawa et al., 1998 ), but it is important to be aware that BOLD measures dynamic changes in blood fl ow and is not direc tly linked to neural activity. In newborns, most fMRI stud- ies have focused on the visual system, and have demonstrated that visual activation can be obtained in sedated infants (So uweidane et al., 1999 ). Th e ability to view abnormal decreases in visual activ- ity (Pani grahy and Bluml, 2007 ), asymmetric, or absent activations (Bernal and Altman, 2004 ) makes fMRI a feasible tool to view the visual cortex in disease (Seghier and H ppi, 2010 ). T h is could be extrapolated to other neonatal conditions, possibly including the assessment of pain. Th e non-invasive nature of fMRI allows for repeated scanning in children and long itudinal studies investigat- ing the development of neural networks can be achieved. Investigation of the maturation of EEG responses to peripheral stimuli in infants from 28 w eeks of gestation has shown that these evoked patterns of activity only become dominant aft er approxi- mately 35 weeks of gestation (Fabrizi et al., 2011 ). Th ere appears to be a g radual shift with advancing maturity, from non-specifi c bursts of neuronal activity to mod ality-specifi c activity that is discrimina- tive between noxious and non-noxious events. A further study that has investigated clinical factors that modulate this activity demon- st rates that infants born preterm (24 \200\22332 weeks of gestation) who have reached their d ue date have a larger evoked response following noxious stimulation than normal term-born i nfants (Slater et al., 2010b ). In contrast, there is no diff erence in the magnitude of th e evoked response following non-noxious simulation. Although it cannot be concluded that pr ior pain experience specifi cally alters the subsequent processing of nociceptive informati on, it is clear that some aspect of being born preterm diff erentially alters the cer- ebra l response to nociceptive stimuli compared with non-noxious tactile stimuli. Recent magneti c resonance imaging (MRI) data supports this observation and suggests that early procedural pain in very preterm infants contributes to impaired brain development (Brummelte et al., 2012 ; see also Grunau, Chapter 4, this volume). It is possible that the anatomical alterat ions observed in infants exposed to excessive nociceptive inputs may underpin some of the f unctional diff erences in subsequent nociceptive processing. In the MRI scanner, neonates and infants are asleep, either natu- rally or with sedation, a nd this is one of the major limitations of fMRI \200\224due to the lack of cooperation, tas ks involving active par- ticipation cannot be performed (Souweidane et al., 1999 ). However , even asleep, infants show distinct brain activations to cognitive (Redcay et al., 2008 ) and sensory (Seghier et al., 2006 ) passive tasks. A more recent discovery is the fl uctuat ion in BOLD fMRI response to intrinsic neuronal activity (Fox and Raichle, 2007 ). Collecti ng fMRI data during periods of quiet rest (i.e. without any task) have revealed strong anat omical and functional connections forming a resting-state network (RSN) in the absence of e xternal stimuli (Raichle and Snyder, 2007 ). Resting-states in infants born preterm or at t erm have found fi ve consistent RSN cortical \200\230hubs \200\231: the pri- mary visual c ortex; bilateral sensorimotor areas; bilateral auditory cortex; a network including the pre cuneus area, lateral parietal cor- tex, and the cerebellum; as well as an anterior network that incor- porated the medial and dorsolateral prefrontal cortex (Fransson et al., 2007 ). With resting-state networks being identifi ed in healthy infants there is a growing intere st in evaluating network alterations in infants with brain injury (Seghier and H ppi, 20 10 ). Magnetic resonance imaging MRI has revolutionized the way we approach pain research. Being non-invasive and non-ionizing, MRI is the ideal technique to inves- tigate the anatomical ( e.g. structure and maturation) and functional (e.g. activity and connectivity) networks of the brain involved in the CNS processing of sensory stimulation during early human developm ent. In neonates, there is great potential for the various MRI tech- niques to elucidate the hig her CNS processing of noxious stimuli. RSNs in healthy neonates do not entirely correspond to those com- monly found in adults (Fransson et al., 2011 ). Assuming that the cortical hu bs denote areas involved in tasks requiring high infor- mation processing (Sporns et al., 2 007 ) some reorganization of RSNs must occur between birth and adulthood. It is proposed th at cortical hubs in infants may be dominated by processes involving Th e transient subplate zone (Kostovic and Molliver, 1974 ) is one of the main signs of cor tical immaturity (Kostovic and Vasung, 2009 ) and is signifi cant for the normal developmen t of the human SECTION 5 measurement of pain 404 refl exive behavior, and those in adults centered toward complex, adaptive behavior (Franss on et al., 2011 ). Any measure of activity in the CNS following a noxious input in non-verbal patients, be it behavioral, autonomical, or neurophysi- ological is ultimately a surrogate measure of the e xperience and cannot tell us what an infant feels. Nevertheless, direct measure- ment of br ain activity may further our understanding of how nox- ious events are processed by the inf ant nervous system. While MRI has been extensively used in adult pain studies, and spe- cifi c network disrupti ons have been described in several adult pathol- ogies including chronic back pain (Baliki et al., 2008 ), its application in children has only recently begun and to date has not bee n used to advance our understanding of infant pain. In adults, pain studies uti- lizing fMR I have involved healthy volunteers or patients. Despite the variability in individual exper ience, a central \200\230pain matrix \200\231 has been described with some areas of the br ain showing consistent patterns of activation following noxious stimulation (Seifert and Ma ihofner, 2009 ). Th ese brain areas include, but are not limited to, the thala- mus, primar y somatosensory cortex (S1), secondary somatosensory cortex (S2), insula, forebrain, and an terior cingulate cortex (ACC) (Tracey and Mantyh, 2007 ) and involves areas traditionally i nvolved in modulating the cognitive (Apkarian et al., 2004 ) and emotional (Becerra et al., 2001 ) aspects of pain perception. References Alexander , A. L. , Lee , J. E. , Lazar , M. , and Field , A. S. ( 2007 ). Diff usion tensor imaging of the brain . Neurotherapeutics , 4 , 316 \200\223329. Andr , M. , Lamblin , M. D. , d \200\231Allest , A. M. , Curzi-Dascalova , L. , Moussall i- Salefranque , F. , Nguyen Th e Tich , S. , et al . ( 2010 ). Electroencephalography in p remature and full-term infants. Developmental features and glossary . Neurophysiol Clin , 4 0 (2), 59 \200\223124. Anand , K. J. , Sippell , W. G. , and Aynsley-Green , A. ( 1987 ). Randomized trial of fent anyl anesthesia in preterm babies undergoing surgery: eff ects on the stress response . Lan cet , 1 (8527), 243 \200\223248. Andrews , K. A. , and Fitzgerald , M. ( 1994 ). Th e cutaneous withdrawal refl ex in human neonates: sensitization, receptive fi elds, and the eff ects of contralateral stimulation . Pain , 56 , 95 \200\223101. To date, there is only one MRI study investigating the processing of noxious stimuli in chi ldren (Hohmeister et al., 2010 ). Cerebral pain response in children and adolescents aged 1 1 to16 years were compared in children with experience of a neonatal intensive care unit (N ICU) born preterm or at term, and term born children without NICU experience. Preterm but n ot term NICU children displayed an exaggerated noxious-specifi c response compared with con trols when stimulated with moderately painful heat stimuli. Brain regions, which classicall y form part of the adult pain matrix, were activated during noxious stimulation but were no t observed during non-painful warmth stimulation. Further, ex-preterm chil- dren displayed an increased sensitization to painful stimuli with- out habituation. Th is would suggest th at in the long term, previous experience of NICU might cause neurodevelopmental changes and cause an alteration in some aspects of nociceptive processing (see also Hermann, Chapter 4 0 , this volume). Andrews , K. A. , and Fitzgerald , M. ( 1997 ). A comparison of the mechanically and electr ically evoked fl exion refl ex as a quantifi able measure of responses to innocuous and nox ious skin stimulation in human neonates . J Physiology , 501.P , 98P . Andrews , K. A. , and Fitzgerald , M. ( 1999 ). Th e cutaneous fl exion refl ex in human ne onates: a quantitative study of threshold and stimulus/ response characteristics, following single and repeated stimuli . Dev Med Child Neurol , 41 , 696 \200\223703. Apkarian , A. V. , Sosa , Y. , Krauss , B. R. , Th omas , P. S. , Fredrickson , B. E. , Lev y , R. E. , et al . ( 2004 ). Chronic pain patients are impaired on an emotional decision-m aking task . Pain , 108 , 129 \200\223136. Baliki , M. N. , Geha , P. Y. , Apkarian , A. V. , and Chialvo , D. R. ( 2008 ). Beyond fee ling: chronic pain hurts the brain, disrupting the default-mode network dynamics . J Neuros ci , 28 , 1398 \200\2231403. Bartocci , M. , Bergqvist , L. L. , Lagercrantz , H. , and Anand , K. J. ( 2006 ). Pain act ivates cortical areas in the preterm newborn brain . Pain , 122 (1 \200\2232), 109 \200 \223117. A recent imaging development may begin to disclose the reor- ganization of cortical hubs. D iff usion tensor imaging (DTI) makes it possible to visualize the connections between diff erent brain regions by measuring changes in the diff usion of water in white matter tracts (Alexander et al., 2007 ). DTI can identify diff er- ences between normal and abnormal trac ts, and when combined with fMRI studies can help understand the structural connectivity bet ween diff erent brain regions (H ppi and Dubois, 2006 ). With resting-state functional c onnectivity being based, but not exclu- sively, on direct anatomical connections (Damoiseau x and Greicius, 2009 ), the assessment of the functional and structural relationships in th e infant brain may begin to be investigated. Becerra , L. , Breiter , H. C. , Wise , R. , Gonzalez , R. G. , and Borsook , D. ( 2001 ). Reward circuitry activation by noxious thermal stimuli . Neuron , 32 , 927 \200\223946. Bernal , B. and Altman , N. ( 2004 ). Visual functional magnetic resonance imaging in patie nts with Sturge-Weber syndrome . 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C. ( 1977 ). Comparative study of perceived pain and nociceptive fl exion refl ex in man . Pain , 3 , 69 \200\22380. CHAPTER 40 Sensory processing and neurophysiological evaluation in children Christiane Herm ann Summary Traditionally, pain in children is assessed by behavioral observa- tion and by self -report with a focus on perceived pain intensity. In this chapter, more recently developed standardized psychophysical procedures to assess abnormalities in sensory and pain processi ng and non-invasive neurophysiological methods to unravel the neu- ronal mechanisms underly ing the experience of pain are described and how they have been used for investigating pedi atric pain. For both sensory and neurophysiological assessment methods, the underlying assu mptions and theoretical underpinnings are outlined. Following a brief overview of commonly used meas- ures and methods, methodological issues are addressed that need to be considered when planning to use such methods and when interpreting the obtained fi ndings. Finally, e mpirical examples are reviewed that highlight the use of these techniques for addressing ma jor research questions such as the development of pain process- ing and changes in sensory and pain processing associated with chronic pediatric pain. fi ndings have been related to specifi c neuronal mechanisms, such interpretations remain p utative as the neuronal substrate is not directly assessed. For investigating neuronal mechanisms of pain, many neurophys- iological methods ranging fr om single-cell or nerve fi bre recordings to neuroimaging are available. Here, the focus li es on non-invasive neuroimaging methods such as near-infrared spectroscopy (NIRS), electroe ncephalography (EEG), and functional magnetic resonance imaging (fMRI) which have been used for studying pediatric pain. Neurophysiological methods for the diagnosis of disease-relat ed pathology will not be addressed. Quantitative sensory testing Over the past 25 years, quantitative sensory testing (QST) has been developed as a method to systematically assess the functioning of sensory and pain pa thways. Interest in QST originally grew from eff orts for a better diagnosis and mechanism- based understanding of neuropathic pain. Neuropathic pain is of neurogenic origin and arise s as a direct consequence of lesions or diseases of the soma- tosensory system (Treede et a l., 2008 ; see Walker Chapter 21, this volume). It is characterized by various symptoms and signs such as sensory loss, paraesthesias, spontaneous onging or paroxysmal pain, and stim ulus-evoked pain presumably resulting from patho- physiological somatosensory functioning. Th e traditional clinical exam of these symptoms has limitations as it is mostly qualitativ e in nature and lacks standardization of stimulation and measurement (Hansson et al., 2007 ). Introduction Th e International Association for the Study of Pain (IASP) defi nes pain \200\230as an unpleasant sensory and emotional experience associ- ated with actual or poten tial tissue damage, or described in terms of such damage \200\231 (Merskey and Bogduk, 1994 , p. 210). Accordingly, sensory processing represents a core component of the pain experi- ence. Pain needs to be distinguished from nociception. Nociception entails all physiologic al processes involved in the transduction of nociceptive (i.e. noxious or potentially tissu e-damaging) input by receptors, and the peripheral and central neuronal activity involved i n the subsequent processing of nociceptive input. By contrast, pain is a complex subjective experience that is correlated with nocic- eptive activity, but is not fully accounted for by it. Dating back to the nineteenth century, psychophysics has evolved as the scientifi c study of the relationship between physical stimuli (e.g. visual, audi- tory) and the subjec tive perception they induce. For the sensory evaluation in pain states, a variety of psycho physical methods, oft en referred to as quantitative sensory testing, have been developed t hat allow quantifi cation of an individual \200\231s subjective response to pain- ful and n on-painful stimuli. Although abnormal psychophysical QST has been derived from experimental psychophysical meth- ods. Traditionally, psychophysi cal procedures can be divided into two broad classes depending on whether it is the stimulu s or the response that is the primary dependent variable (Gracely, 2006 ). Response-depende nt methods assess individuals \200\231 variable responses to a predefi ned set of stimuli. Examples are almost all methods used for measuring suprathreshold pain such as rating scale s. Stimulus- dependent psychophysical methods entail the administration of varying stimuli with a fi xed response and it is stimulus intensity that is measured. Typically, such stimu lus-dependent methods are used for determining thresholds. Yet, they also allow suprathresh - old pain assessment such as pain tolerance. For determining thresh- olds, three classical psychophysical procedures are established. SECTION 5 measurement of pain 408 Method of limits (MLI): this method entails the administration of stimuli of varying intens ity until the subject notes a certain sensa- tion and indicates this verbally or behavioura lly. Usually, stimuli are delivered in ascending series, the subject \200\231s response sto ps fur- ther increase in intensity. Sensory detection and pain thresholds are defi ned as t he stimulus intensity required to evoke a given sen- sation (e.g. touch, warmth) or pain, r espectively. Whereas sensory detection thresholds rely on discriminating the presence versu s absence of a specifi c sensation, pain thresholds are not only infl u- enced by perceptua l sensitivity, but also by the given verbal label and the subject \200\231s pain labeling b ehavior and the ensuing response bias (Gracely, 2006 ). In order to reduce expectancy or ha bituation eff ects, it is oft en recommended to use both ascending and descend- ing series. MLI is reaction-time dependent (Yarnitsky and Ochoa, 1990 ). Most likely, this accounts fo r thermal thresholds being about 1.5 C higher when determined by MLI as compared to reac tion- time independent methods (e.g. Defrin et al., 2006 ; children: Meier et al., 2001 ). Despite this limitation, MLI is frequently used because instructions are simple and it is l ess time-consuming than other procedures. Th ere are reaction time-independent MLI variants . Th e method of levels entails the administration of stimuli with pre- defi ned intensity and the subject \200\231s response is recorded. Depending on the subject \200\231s positive or negative response, stimulus intensity is systematically either decreased (following a p ositive response) or increased (following a negative response) until it converges onto the threshold (Meier et al., 2001 ; Yarnitsky et al., 1990 ). Stimulus intensity can either be changed in absolute steps or in steps of modality-specifi c, just noticeable, diff erences (Yarnitsky, 1997 ). Reaction-time independent measures are more reliable than reac- tion-ti me dependent measures, but require more trials (Yarnitsky, 1997 ). is helpful for detecting sensory losses and gains such as altered pain sensitivity (hyperal gesia, allodynia, hyperpathia). Including suprathreshold stimuli is important because hyper sensitivity can be observed if thresholds are reduced, elevated, or unchanged. \200\230Dyn amic \200\231 measures require activation of the pain system such that possible changes in specifi c mechanisms of pain processing become evident. For example, sensitization to nocic eptive input is a key factor in the development of chronic pain. Such sensitization occurs in the peripheral and the central nervous system (Woolf, 2011 ). In humans, temporal summat ion (i.e. the increase in per- ceived pain intensity upon prolonged or repetitive painful s timula- tion), is considered to represent a perceptual correlate primarily of central sensi tization (e.g. Granot et al., 2006 ). Accordingly, some QST protocols include measures of t emporal summation. Th e test- ing of descending pain inhibitory mechanisms using procedures of painful counter-irritation is another example of a dynamic QST measure. Comprehensive QST protocols usually include the testing of diff erent sensory modalities su ch as thermal, touch, pressure, and vibratory stimuli. For obvious reasons of impracticabil ity and problems of standardization, chemical stimulation is not included. Testing diff ere nt sensory modalities allows the assessment of the integrity of diff erent types of recepto rs, peripheral nerve fi bres and information integration within the central nervous system. Mechanical stimuli such as light touch, pressure, or punctate stimuli activate A \237 , A , and C fi bres (see Table 40.1 ; Walk et al., 2009 ). Th e processing of warm and heat stimuli is dependent on C-fi bre activa- tion, cold sensations and noxious cold involve A -fi bre and C-fi bre activity, respectively. Th e processing of painful stimuli is media ted both by modality-specifi c and convergent pathways (Craig, 2003 ). Hence, pain sensatio n is the result of central integration of sensory non-nociceptive information and nocicepto r input (Defrin et al., 2002 ). Accordingly, correlations between (supra-)threshold meas- u res across modalities are typically low (e.g. Bhalang et al., 2005 ). Indeed, factor analyz es of diff erent pain modality measures (e.g. heat, ischaemic, pressure, electrical) reveal ed separate factors each representing a sensory modality (Hastie et al., 2005 ; Neziri et a l., 2011a ). In addition, measures of temporal summation loaded on a separate factor, suppo rting the distinction between static and dynamic measures (Hastie et al., 2005 ). Method of constant stimuli: similar to MLI, the subject is exposed to a set of stimuli with predefi ned intensity above and below the threshold of interest. However, these stimuli ar e presented ran- domly and the subject is asked to indicate whether or not the stimulus evo ked a certain sensation. Th e threshold is defi ned as the intensity that the subject repor ts as the sensation of interest in 50% of the trials. Method of adjustment: unlike MLI, this method requires the sub- ject to directly adjust sti mulus intensity until a certain sensation occurs. Th is procedure has to be repeated many t imes. Aside from the long duration, it is disadvantageous in that some of the stimula- tion devices for certain pain modalities do not allow the subject to control stimulus intensity . Sensory testing site When performing QST, the choice of testing sites is important. Th e ob tained values vary considerably between diff erent body regions (e.g. Defrin et al., 2006 ; children: Hogeweg et al., 1996 ; Meier et al., 2001 ; Meh and Denislic, 1994 ). In one of the most comprehensive studies in healthy adults, QST was performed bilaterally on the face , and at the hand and foot (Rolke et al., 2006b ). Similarly, in children, while no left \200\223right diff erences were observed, sensitivity was highest in the face and lowest in the foot and, with interme- diate values in the hand (Blankenburg et al., 2010 ). Presumab ly, this region- specifi c sensitivity is accounted for by diff erences in innervation and/ or thickness of the epidermis. However, these diff erences, at least for thresholds obtaine d by MLI, may also be a result of reaction-time-dependent threshold determination. If the m ethod of levels rather than MLI was used, diff erences in sensitiv- ity between body region s such as foot versus upper body were no longer observed, at least for thermal stimuli (Def rin et al., 2006 ). Although, in healthy subjects, thermal pain sensitivity does not In most cases, QST protocols exclusively rely on MLI for deter- mining sensory detection an d pain thresholds; mostly for practi- cal reasons, such as keeping the duration of the test ing within reasonable limits. Length of testing is an issue because the subject should be a lert and awake and motivated to cooperate with the experimenter. QST measures Sensory modalities Quantitative sensory testing does not refer to defi ned set of meas- ures. QST measures can broadly be categorized as static or dynamic (Arendt-Nielse n and Yarnitsky, 2009 ). Th reshold measures (e.g. detection, pain) and pain magnitude rati ngs for a given stimulus are typically considered as \200\230static \200\231 and are used for assessing the basal state of the somatosensory system. Th reshold determination CHAPTER 40 sensory processing and neurophysiological evaluation in children 409