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CHAPTER 39 Th e neurophysiological evaluation of nociceptive responses in neonates Ravi Poo run and Rebeccah Slater Summary Human pain and suff ering is most oft en communicated by language. Th e ability to communicate verbally puts adults and older children at a distinct advantage over our young counterparts. Infants and newborns cannot talk \200\224which makes the reliable subjective meas- urement of pain diffi cult in this population. In neonates, we have traditionally rel ied on objective measurements of physiological and behavioral responses to noxious stimulat ion (Stevens et al., 2007 ), and research continues to search for the best measures of pain in neonates (Stevens and Franck, 2001 ; see also Lee and Stevens, Chapter 35 , this volume ). process noxious stimuli, and that this information is transmitted to the brain. However, wh ile the use of neurophysiological and neuroimaging techniques has been extensively used in the study of adult pain, these techniques have had relatively limited use in eval- uating t he response to nociceptive stimuli in neonates and infants. Neurophysiological measures in older children are discussed by Hermann ( Chapter 40 , this volume). Key stages of anatomical development have been identifi ed in the human infant brain, such as: \231 Th e growth of thalamocortical axons into the cortical plate from 26 gestational weeks (Kostovic and Judas, 2002 ). In this chapter we review the use of quantitative neurophysio- logical techniques to evalua te nociceptive responses in the central nervous system of the neonate. We will discuss how: \231 Th e elaboration of terminals and onset of cortical diff erentiation from 31 weeks. \231 Th e formation of callosal and association pathways from 34 \231 electromyography (EMG) can be used to measure fl exion refl ex weeks (Kostovic and Judas, 2010 ). withdrawal. However, almost nothing is known about the functional cor- relates of these events. Th roug h investigation of the developing human nervous system we can try to ask \200\230What is t he pattern of brain activity that underpins the human experience of pain? \200\231. In the adult brain, numerous structures are involved in the experience of pain, including the brai nstem, thalamus, and a rich variety of cortical areas, which includes the somatosensory cor tex, cingulate cortex, insula, and amygdale (Tracey and Mantyh, 2007 ). However, the extent to which these structures are involved in infant pain is unknown. Direct measurement of br ain activity, using a variety of techniques, will further our understanding of how noxious events are processed by the infant nervous system. In this chapter we will highlight some o f the important advances that have been made using neurophysiological measures to study res ponses to noxious and non-noxious stimuli in early life. \231 near-infrared spectroscopy (NIRS) and electroencephalography (EEG) can be used to me asure brain activity evoked by noxious stimulation. \231 magnetic resonance imaging (MRI) may be exploited to investi- gate the development o f nociceptive processing in the neonatal and infant brain. Th e appropriate use of these techniques may help us understand how the functional developm ent of the human brain enables processing of nociceptive stimuli. Introduction It was once a long-held belief that due to the immaturity of the peripheral an d central nervous systems, neonates did not feel pain. Coupled with limited data regarding the safety and effi cacy of anal- gesia in neonates, minimal analgesia was oft en administe red for procedures that would most certainly be painful in adults (Anand et al., 1987 ). Ov er the past 30 years, the study of childhood and infant pain has evolved to become a subjec t of active research (McGrath and Unruh, 2002 ). It is now accepted that human infants Electromyography Electromyography (EMG) records the electrical activity produced by skeleta l muscle. In neonates, surface EMG is preferred over the more-invasive intramuscular EMG te chniques, and is most oft en used to monitor the fl exion refl ex withdrawal activity of th e SECTION 5 measurement of pain 402 underlying biceps femoris in the posterior thigh, or tibialis ante- rior in the anterolater al lower leg. Th e fl exion withdrawal refl ex (Sherrington, 1910 ) has been used in adult subjects to study the spi- nal processing of nociceptive stimuli (Willer, 1977 ) and analge sic sensitivity (Willer, 1985 ). In both preterm and term neonates, the fl exion refl ex ca n be evoked by mechanical or electrical stimulation (Andrews and Fitzgerald, 1994 , 1997 , 1999 ; Fitzgerald et al., 1989 ). However, the stimulus intensity or threshold or for elici ting refl ex activity is lower than in adults (Hugon, 1973 ), and refl exes can be elicited by non-painful stimuli. Increasing age sees a gradual reor- ganization and decrease in spi nal cord excitability (Jennings and Fitzgerald, 1996 , 1998 ; see also Walker and Baccei, C hapter 6, this volume), which is accompanied by a rise in threshold of the neonatal withdra wal refl ex (Andrews and Fitzgerald, 1994 ; Fitzgerald et al., 1988 ). Below 35 weeks postc onceptual age, preterm infants display sensitization to innocuous stimuli and a reduction i n threshold (Andrews and Fitzgerald, 1994 ) whilst older infants display habitu- ation to t he same non-noxious stimuli. Habituation is character- ized by a reduction in the number of observed refl ex withdrawal responses but with no alteration in withdrawal threshold follo w- ing repeated stimulation (Andrews and Fitzgerald, 1994 ; Fitzgerald et al., 1988 ). Simi larly, habituation of refl ex responses can be seen in adults with intact or divided spinal cords (Dimitrijevi \207 and Nathan, 1970 ; Dimitrijevi \207 et al., 1972 ). somatosensory cortex in preterm neonates from approximately 25 weeks of gestation. Th e mag nitude of this activity is dependent on the infant \200\231s age, but is also infl uenced b y other clinical features, such as the sleep state of the infants. Similar changes in hemog lobin concentration in the somatosensory cortex were not found when non-skin-breaking tacti le stimuli were applied to the surface of the skin, even though refl ex withdrawal response s were observed, sug- gesting the observed changes in brain activity were related to the no xious input. Using NIRS for clinical bedside pain assessment remains chal- lenging because movement arti facts can interfere with the quality of the recorded signal and there is an extremely high attrition rate for recording of high-quality data. However, with secure optode placement, m ovement artifacts can be minimized, and careful con- trol of the infants \200\231 environme nt can improve the quality and accu- racy of the recordings. Further NIRS studies have demo nstrated that following noxious stimuli there is a good correlation between the magnitude o f the haemodynamic changes recorded in the con- tralateral somatosensory cortex and the mor e classically recorded change in neonatal facial expression (Slater et al., 2009 ). Other e xperimental and clinical conditions have also been shown to alter the size of the haemodyna mic response, such as the location of the stimulus and the number of prior painful procedur es (Ozawa et al., 2010 , 2011 ; Slater et al., 2006 ). Importantly, all these studies lead to the conclusion that infants who are exposed to noxious stimuli dur- ing this immature de velopmental period, process this information at the level of the cortex, even though some i nfants do not manifest an observable behavioral response (Slater et al., 2008 ). Over the postnatal period, somatosensory and motor pathways in the central nervous system ( CNS) are continually maturing (Fitzgerald, 2005 ). While EMG techniques cannot tell us anyt hing about the subjective experience or perception of pain, they are extremely useful in un derstanding the development of spinal noci- ceptive processing. Th ese studies provide some evidence that preterm and newborn infant responses to noxious s timulation are not purely refl exive, but the information is transmitted to the infant cort ex. While this does not provide evidence of the perception of pain and cannot tell us what an infant feels, it is likely that activity in higher-level cortical regions of the brain i s necessary, though maybe not suffi cient, for the perception of pain. Near-infrared spectroscopy Near-infrared spectroscopy (NIRS) can be used to measure blood a nd tissue oxygenation. Th e method is based on spectroscopic measurements of oxygenated and deoxygenated hemoglobin con- centration and depends on the transparency of biological tiss ue to near-infrared light. It uses the optical property that the absorption of near-infrare d light is dependent on the amount of oxygenated and deoxygenated hemoglobin present in the tissue (Owen-Reece et al., 1999 ). Source and detector pairs can be placed on the sur- fac e of the skull and local changes in the intensity of the emitted and absorbed light can qua ntify changes in tissue oxygenation. In addition, absolute parameters such as blood fl ow a nd cerebral blood volume can be calculated. Th e measurement of changes in haemodynamic act ivity \200\224as a proxy measure of functional brain activity \200\224is based on the assum ption that increased tissue oxygena- tion represents an increase in regional cerebral blood fl ow, asso- ciated with an increase in underlying neural activity (Logothetis et al., 200 1 ). 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.