Kehlet, H. and Rathmell, J. P. (2010). Persistent postsurgical pain: the path forward throu gh better design of clinical studies. Anesthesiology, 112 , 514 \200\223515 . Kissin, I. (2000). Preemptive analgesia. Anesthesiology, 93 , 1138 \200\2231143 . Kleiman, V., Clarke, H., and Katz, J. (2011). Sensitivity to pain traumatization: a higher-order fac tor underlying pain-related anxiety, pain catastrophizing and anxiety sensitivity among pat ients scheduled for major surgery. Pain Res Manag, 16 , 169 \200\223177 . Minuchin, S., Baker, L., Rosman, B. L., Liebman, R., Milman, L., and Todd, T. C. (1975). A conceptual model of psychosomatic illness in children. Family organization and family thera py. Arch Gen Psychiatry, 32 , 1031 \200\2231038 . Ong, C. K., Lirk, P., Seymour, R. A., and Jenkins, B. J. (2005). Th e effi cacy of preempti ve analgesia for acute postoperative pain management: a meta- analysis. Anesth Analg, 100 , 757 \200\223773 . Konijnenberg, A. Y., Uiterwaal, C. S., Kimpen, J. L., Van Der Hoeven, J., Buitelaar, J. K., and De Graeff -Meeder, E. R. (2005). Children with unexplained chronic pain: substantial i mpairment in everyday life. Arch Dis Child, 90 , 680 \200\223686 . Ozcengiz, D., Gunduz, M., Ozbek, H., and Isik, G. (2001). Comparison of caudal morphine and tramadol for postoperative pain control in children undergoing inguinal herniorrhaphy. Pae diatr Anaesth, 11 , 459 \200\223464 . Pag , M. G., Stinson, J., Campbell, F., Isaac, L., and Katz, J. (2013). Identifi cation of pain-related psychological risk factors for the de velopment and maintenance of chronic pediatric post-surgical pain. J Pain Res , 6, 167 \200 \223180. Kovacs, F. M., Gestoso, M., Gil Del Real, M. T., Lopez, J., Mufraggi, N., and Mendez, J. I. (2003). Risk factors for non-specifi c low back pain in schoolchildren and their parents: a population based study. Pain, 103 , 259 \200\223268 . Kraemer, H. C., Kazdin, A. E., Off ord, D. R., Kessler, R. C., Jensen, P. S., and Kupfer, D . J. (1997). Coming to terms with the terms of risk. Arch Gen Psychiatry, 54 , 337 \200\223 343 . Palermo, T. M. and Chambers, C. T. (2005). Parent and family factors in pediatric chronic p ain and disability: an integrative approach. Pain, 119 , 1 \200\2234 . Kroner-Herwig, B. (2011). Psychological treatments for pediatric headache. Expert Rev Neurother, 11 , 403 \200\223410 . Palermo, T. M., Witherspoon, D., Valenzuela, D., and Drotar, D. D. (2004). Development and validation of the Child Activity Limitations Interview: a measure of pain-related functiona l impairment in school-age children and adolescents. Pain, 109 , 461 \200\22370 . Kundra, P., Deepalak Shmi, K., and Ravishankar, M. (1998). Preemptive caudal bupivicaine an d morphine for postoperative analgesia in children. Anesth Analg, 87 , 52 \200\22356 . Lamontagne, L. L., Hepworth, J. T., Cohen, F., and Salisbury, M. H. (2003). Cognitive-behav ioral intervention eff ects on adolescents \200\231 anxiety and pain following spinal fusio n surgery. Nurs Res, 52 , 183 \200\223190 . Parry, S. (2011). Acute pain management in the neonate. Anaesth Intensive Care, 12 , 121 \200\223125 . CHAPTER 5 prevention of the development and maintenance 49 Turk, D. C. (2002). A diathesis-stress model of chronic pain and disability Perquin, C. W., Hunfeld, J. A., Hazebroek-Kampschreur, A. A., Van Suijlekom-Smit, L. W., Pa sschier, J., Koes, B. W., et al . (2003). Th e natural course of chronic benign pain in chi ldhood and adolescence: a two-year population-based follow-up study. Eur J Pain, 7 , 551 \200\223559 . following traumatic injury. Pain Res Manag, 7 , 9 \200\22319 . Turk, D. C., Dworkin, R. H., Allen, R. R., Bellamy, N., Brandenburg, N., Carr, D. B., et al . (2003). Core outcome domains for chronic pain clinical trials: IMMPACT recommendations. Pain, 106 , 337 \200\223345 . Pillai Riddell, R. R., Racine, N. M., Turcotte, K., Uman, L. S., Horton, R. E., Din Osmun, L., et al . 2011. Non-pharmacological management of infant and young child procedural pain. Cochrane Database Syst Rev , 10, CD006275. Turk, D. C., Dworkin, R. H., Revicki, D., Harding, G., Burke, L. B., Cella, D., et al . (20 08). Identifying important outcome domains for chronic pain clinical trials: an IMMPACT sur vey of people with pain. Pain, 137 , 276 \200\223285 . Priest, T. D. and Hoggart, B. (2002). Chronic pain: mechanisms and treatment. Curr Opin Pharmacol, 2 , 310 \200\223315 . Uman, L. S., Chambers, C. T., McGrath, P. J., and Kisely, S. (2006). Psychological interven tions for needle-related procedural pain and distress in children and adolescents. Cochrane Database Syst Rev , 4, CD005179 . Prins, Y., Crous, L., and Louw, Q. A. (2008). A systematic review of posture and psychosoci al factors as contributors to upper quadrant musculoskeletal pain in children and adolescen ts. Physiother Th eory Pract, 24 , 221 \200\223242 . Sawyer, M. G., Carbone, J. A., Whitham , J. N., Roberton, D. M., Taplin, J. E., Varni, J. W., et al . (2005). Th e relationship be tween health-related quality of life, pain, and coping strategies in juvenile arthritis \200\224a one year prospective study. Qual Life Res, 14 , 1585 \200\2231598 . Van Slyke, D. A. and Walker, L. S. (2006). Mothers \200\231 responses to children \200\231s pain. Clin J Pain, 22 , 387 \200\223391 . Varni, J. W., Stucky, B. D., Th issen, D., Dewitt, E. M., Irwin, D. E., Lai, J. S., et al . (2010). PROMIS Pediatric Pain Interference Scale: an item response theory analysis of the pediatric pain item bank. J Pain, 11 , 1109 \200\2231119 . Vervoort, T., Goubert, L., Eccle ston, C., Bijttebier, P., and Crombez, G. (2006). Catastrophic thinking about pain is indep endently associated with pain severity, disability, and somatic complaints in school childr en and children with chronic pain. J Pediatr Psychol, 31 , 674 \200\223683 . Scharff , L. (1997). Recurrent abdominal pain in children: a review of psychological factor s and treatment. Clin Psychol Rev, 17 , 145 \200\223166 . Schechter, N. L. (2008). From the ouchless place to comfort central: the evolution of a concept. Pediatrics, 122 (Suppl 3), S154 \200\223160 . Schulte, I. E., Petermann, F., and Noeker, M. (2010). Functional abdominal pain in childhoo d: from etiology to maladaptation. Psychother Psychosom, 79 , 73 \200\22386 . Victor, S. and Ryan, S. W. (2003). Drugs for preventing migraine headaches in children. Cochrane Database Syst Rev , 4, CD002761. Vikat, A., Rimpela, M., Salminen, J. J., Rimpela, A., Savolainen, A., and Virtanen, S. M. ( 2000). Neck or shoulder pain and low back pain in Finnish adolescents. Scand J Public Healt h, 28 , 164 \200\223173 . Seebach, C. L., Kirkhart, M., Lating, J. M., Wegener, S. T., Song, Y., Riley, L. H., 3rd et al . (2012). Examining the role of positive and negative aff ect in recovery from spine su rgery. Pain, 153 , 518 \200\223525 . Vlaeyen, J. W., Kole-Snijders, A. M., Boeren, R. G., and Van Eek, H. (1995). Fear of moveme nt/(re)injury in chronic low back pain and its relation to behavioral performance. Pain, 62 , 363 \200\223372 . Simons, L. E., Sieberg, C. B., Carpino, E., Logan, D., and Berde, C. (2011). Th e Fear of P ain Questionnaire (FOPQ): assessment of pain-related fear among children and adolescents wi th chronic pain. J Pain, 12 , 677 \200\223686 . Sjolie, A. N. (2002). Psychosocial correlat es of low-back pain in adolescents. Vuorimaa, H., Tamm, K., Honkanen, V., Konttinen, Y. T., Komulainen, E., and Santavirta, N. (2008). Empirical classifi cation of children with JIA: a multidimensional approach to pain and well-being. Clin Exp Rheumatol, 26 , 954 \200\223961 . Eur Spine J, 11 , 582 \200\223588 . Stevens, B., Yamada, J., and Ohlsson, A. (2010). Sucrose for analgesia in newborn infants u ndergoing painful procedures. Cochrane Database Syst Rev , 1, CD001069 . Walker, L.S. and Greene, J.W. (1991). Th e functional disability inventory: measuring a neg lected dimension of child health status. J Pediatr Psychol; 16 ; 39 \200\22358 . Taddio, A., Appleton, M., Bortolussi, R., Chambers, C., Dubey, V., Halperin, S., et al . (2 010). Reducing the pain of childhood vaccination: an evidence- based clinical practice guid eline (summary). CMAJ, 182 , 1989 \200\2231995 . Taddio, A., Ilersich, A. L., Ipp, M., Kiku ta, A., and Shah, V. (2009). Physical interventions and injection techniques for reducing i njection pain during routine childhood immunizations: systematic review of randomized contr olled trials and quasi-randomized controlled trials. Clin Th er, 31 Suppl 2, S48 \200\22376 . Walsh, T. M., McGrath, P. J., and Symons, D. K. (2008). Attachment dimensions and young chi ldren \200\231s response to pain. Pain Res Manag, 13 , 33 \200\22340 . Wicksell, R. K., Melin, L., Lekander, M., and Olsson, G. L. (2009). Evaluating the eff ecti veness of exposure and acceptance strategies to improve functioning and quality of life in longstanding pediatric pain \200\224a randomized controlled trial. Pain, 141 , 248 \200\223 257 . Tsao, J. C., Evans, S., Meldrum, M., and Zeltzer, L. K. (2009). Sex diff erences in anxiety sensitivity among children with chronic pain and non-clinical children. J Pain Manag, 2 , 151 \200\223161 . Wilson, A. C., Lewandowski, A. S., and Palermo, T. M. (2011). Fear-avoidance beliefs and pa rental responses to pain in adolescents with chronic pain. Pain Res Manag, 16 , 178 \200 \223282 . Tsao, J. C. and Zeltzer, L. K. (2005). Complementary and alternative medicine approaches fo r pediatric pain: a review of the state-of-the-science. Evid Based Complement Alternat Med, 2 , 149 \200\223159 . Yaster, M. (2010). Multimodal analgesia in children. Eur J Anaesthesiol, 27 , 851 \200\223857 . Turk, D. C. (1996). Biopsychosocial perspective on chronic pain. In R. J. Gatchel and D. C. Turk (eds) Psychological approaches to pain management: a practitioner \200\231s handbook , pp. 6 \200\22323. New York : Guilford Press . This page intentionally left blank SECTION 2 Biological basis of pediatric pain This page intentionally left blank CHAPTER 6 Nociceptive signaling in the periphery and spinal cord Suellen M. Walker and Mark L. Baccei Summary Responses to painful or noxious stimuli are functional at birth. However, postnatal changes in the transmitters, receptors, and pathways involved in nociceptive signaling res ult in signifi cant age-related changes in the nature and degree of response. from 24 weeks until full-term birth at 40 post-conceptional weeks (Fitzgerald, 1991a). Pups mature relatively rapidly and can be con- sidered infants by postnatal day (P) 7, and as c hildren or young adolescents when they are weaned at P21. Rather than represent- ing absolu te correlations, these timelines provide a framework for assessing progressive changes in f unction throughout development, and it is important to note that diff erent regions of the nervous sys- tem mature at diff erent rates. Subsequent chapters will extend this discussio n to: interactions between neurons and immune cells in nociceptive processing (Vega-Avelair a and Beggs, Chapter 7) and transmission to higher centers and modulation by brainstem cen- tres (Fitzgerald, Chapter 8) (Figure 6.1 ). Visceral pain processing is discussed by Batra et al. (Chapter 27). Noxious mechanical, thermal, and chemical stimuli are detected by peripheral nociceptors, t ransduced into electrical stimuli, and transmitted to the spinal cord. Within the spinal co rd, there are signifi cant postnatal changes in the balance of inhibitory and exci- tatory signaling, that not only infl uence the acute response to aff er- ent input, but can also u nderlie long-term alterations in sensory processing following tissue injury in early life. Evaluating age-related changes in nociceptive signaling is essential not only for understan ding acute behavioral responses to noxious stimuli, but also for identifying the most appro priate and eff ective pain management interventions at diff erent developmental ages. Peripheral nociceptive pathways \200\230Nociception \200\231 refers to the physiological a nd pathophysiological component of sensory processing (which is the focus of the current ch apter), whereas \200\230pain \200\231 encompasses sensory, emotional, and cog- nitive comp onents. However, the terms are oft en used interchange- ably, and will be for the purposes of this chapter. Introduction Th roughout postnatal development, there are signifi cant functional and struc tural changes in the developing nervous system that infl uence nociceptive transmission. Pa tterns of innervation, receptor expression and distribution, and synaptic function within n ociceptive pathways vary with age and infl uence the sensitivity and nature of response. Th is chapter describes the peripheral and spinal cord components of nociceptive processing, and outlines important age-related changes that infl uence the transmission and modulation of pain signaling in early life. Issues related to fetal nociception will not be discussed, as the marked diff erences between in utero and postnatal physiology also infl uence nocic eptive signaling (Mellor et al., 2005). One role of the peripheral nervous system is to sense and trans- duce potentially or actual ly damaging stimuli into electrical signals that are then transmitted along the axon to the central nervous sys- tem (CNS). Peripheral nociceptive pathways have four functional compo nents (Woolf and Ma, 2007): 1. A nociceptor is a high-threshold sensory receptor of the periph- eral somatosensory nerv ous system that is capable of transduc- ing and encoding noxious stimuli. A stimulus is nox ious if it is damaging or threatens damage to normal tissues (Merskey and Bogduk, 1994; see also updated IASP de \201 nitions <http://www. iasp-pain.org/Content/NavigationMenu/Gener alResourceLinks/ PainDe \201 nitions/>). Th e ability to detect and respond to potentially damaging envi- ronmental stimuli is an es sential function that is maintained across species (Smith and Lewin, 2009). Infant rodents provide an estab- lished model for assessment of mammalian development and are frequently u tilized to assess age-dependent changes in nocicep- tive mechanisms, with structural and fu nctional methods that are not possible in clinical studies (Fitzgerald and Walker, 2009). T h e rat pup is born at a relatively immature stage, and data collected from human fetal tis sue and rat pups suggest that the development of peripheral and spinal somatosensory functi on during the fi rst postnatal week in the rat pup corresponds to preterm development 2. A peripheral branch of the axon that conducts the action poten- tial and transports rece ptors/proteins from the cell body to the site of transduction. Two main groups of cutaneous afferents ter- minate as free nerve endings in the epidermis: \231 Myelinated A fi bres associated with high-threshold, slowly adapting mechanorecep tors, and in addition a proportion respond to heat and cold. SECTION 2 biological basis of pediatric pain 54 ASCENDING PATHWAYS DESCENDING PATHWAYS cc cc Hip Hip Po Po ic ic VPM/VPL VMH VPM/VPL VMH Ce Ce PAG DESCENDING MODULATION FROM BRAINSTEM \200 delayed inhibition \200 ? excess facilitation A7 A5 PB LC bc PERIPHERY \200 respond to noxious stimuli \200 peripheral sensitization v RVM Py SPINAL CORD \200 enhanced excitation \200 reduced inhibition DRG REFLEX RESPONSE \200 reduced threshold \200 generalized response Figure 6.1 Diagrammatical representation of major nociceptive pathways. Textboxes highlight di \200 erences during early development. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Neuroscience , Stephen P. Hunt and Patrick W. Mantyh, Th e molecular d ynamics of pain control, Volume 2, Issue 2, p. 85, Copyright 2001, http://www.nature.com /nrn/index.html. \231 Unmyelinated C fi bres, the majority of which are polymodal and respond to noxious m echanical, thermal and chemical stimuli. \200\230Silent \200\231 nociceptors become respon sive to mechanical and/or heat stimuli aft er infl ammation (Smith and Lewin, 2009). \227\206 Electrophysiology/microneurography recordings from sensory aff erent fi bres. Th is can be performed in vitro from isolated skin-nerve preparations (Koltzenburg et al., 199 7; Reeh, 1986), or by in vivo studies in animals and humans (Koltzenburg and Handwerker, 19 94). 3. The neuronal cell body in the dorsal root ganglion (DRG) is the major site for transcrip tion and protein synthesis. In addition to sensory neurons, satellite glial cells are prese nt in the DRG (see Vega-Avelaira and Beggs, Chapter 7, this volume, for further details). T he small cell bodies of nociceptors are divided into two groups (Woolf and Ma, 2007): \227\206 Analysis of receptor function and channel properties in cultured DRG neurons usin g patch clamp recordings and Ca 2+ -imaging techniques. \227\206 Evaluating the organism \200\231s behavior in response to noxious stimuli (e.g. changes in spinal refl ex sensitivity). \231 Th ose that express tyrosine receptor kinase A (TrkA) and respond to nerve growth fa ctor (NGF). Th ese peptidergic nociceptors express calcitonin gene-related peptide (CGRP) a nd substance P (SP) and project to lamina I and lamina II outer in the superfi cial dorsal horn of the spinal cord. An important protective function of the nervous system is its ability to detect and localiz e noxious stimuli and produce rapid refl ex motor responses, which draw the aff ected area away from the potential injury. Evaluating spinal refl ex sensitivity to peripheral stimuli allows quantifi cation of the impact of both injury and/or analgesia (Le Bars et al., 2001 ; Walker et al., 2009). Although thresholds for eliciting refl ex responses vary with age, correlations between stimulus intensity and refl ex response have been demonstrated at all ages in laboratory animals (Holmberg and Schouenborg, 1996), and in human neonates (Andrews and Fitzgerald, 1999) and adults (Willer, 1983). \231 Th ose that express Ret, the transmembrane signaling compo- nent of the receptor for glial cell-line derived neurotrophic fac- tor (GDNF). Th ese non-peptidergic neurons bind isolectin B4 (IB4), express the P2X 3 purine receptor, and project to lamina II inner in th e dorsal horn (Hunt and Mantyh, 2001). 4. The central branch of the axon that projects from the DRG to the super \201 cial dorsal horn and forms synaptic contacts with the second-order neuron. Presynaptic release of neur otransmitter (i.e. glutamate, substance P, brain-derived neurotrophic factor (BDNF)) is reg ulated by modulating calcium in \202 ux in the pri- mary afferent terminal. Peripheral innervation Nociceptive neurons are derived from neural crest cells in the dor- sal part of the neural tube (Fitzgerald, 2005; Woolf and Ma, 2007). During embryonic develo pment, sensory axons extend down the developing limbs to innervate the epidermis (Jackman a nd Fitzgerald, 2000). Larger-diameter A-fi bre innervation occurs fi rst, but by birth in t he rat and by the second trimester in man, both A and C sensory fi bres are distributed to all body regions (Fitzgerald, 2005). In rodent skin, fi bres initially penetrate up to the epidermal Peripheral nociceptive function has been evaluated by multiple experimental techniques, som e of which have been conducted both in laboratory animals and in human experimental or clin ical studies (Smith and Lewin, 2009). Th ese include: CHAPTER 6 nociceptive signaling in the periphery and spinal cord 55 Th ermal and chemical stimuli Transient receptor potential (TRP) ion channels comprise a la rge family of receptors that transduce varying stimuli into electrical potentials (Wang and Woolf, 2005). Receptors expressed in sen- sory neurons or skin which respond in specifi c temperature ranges include TRPV1 to TRPV4, TRPM8, and TRPA1 (Patapoutian et al., 2009). Mes senger RNA (mRNA) levels of the diff erent receptors in the DRG, and neuronal responses to temperature and specifi c chemical ligands, change during postnatal development (Hjerling- Leffl er et al., 2007). surface but then retract to form a subepidermal plexus that becomes more organized over the fi rst 2 postnatal weeks and innervates end organs, such as sweat glands and hair follicle s (Jackman and Fitzgerald, 2000). Navigation of sensory terminals to their target tissue is controlled by long- and short-range guidance cues that permit or inhibit axon growth (i.e. they are attractive or repulsive; Kolodkin and Tessier-Lavigne, 2011). Ephrins are a famil y of cell- surface signaling molecules involved in short-range axon guidance during develop ment that includes class A ephrins interacting with multiple class A ephrin (Eph) receptors . Neural growth cones are inhibited by ephrin-A4, which is localized to the upper epider- m is postnatally, thus restricting skin terminals to lower epidermal regions, and levels decr ease as innervation stabilizes postnatally. However, levels of ephrin-A4 are also infl uenc ed by injury, and downregulation following neonatal full-thickness skin wounds contributes to persistent hyperinnervation (Moss et al., 2005; see Walker, Chapter 3, this volume for f urther details). \227\206 TRPV1 is activated by temperatures greater than 43 C, but also by a range of che micals including capsaicin, and its function is modulated by low pH. Changes in TRPV1 recep tor expression and function, many of which are mediated by NGF, contribute to infl ammatory hyperalgesia. Post-translational changes, which alter channel kinetics, and increased inse rtion of the recep- tor into the membrane, also contribute to increased sensitivity (Linley et al., 2010; Patapoutian et al., 2009). TRPV1 mRNA is present in the DRG prior to and aft er birth, and the receptors are functional as these neurons respond to capsaicin (Hjerling - Leffl er et al., 2007). Th e delivery of TRPV1 to both peripheral and central terminals i s developmentally regulated. TRPV1- positive nerve terminals are present in cutaneous struc tures at P10, but at a reduced density compared with adult animals (Guo et al., 2001) and t he degree of sensitization to capsaicin by NGF is lower (Zhu et al., 2004). Neurotrophins In addition to established roles in modulating nociceptive sensi- tivity in a dulthood (Pezet and McMahon, 2006), neurotrophins regulate multiple aspects of the developm ent of sensory nerves (Fitzgerald, 2005), including: \227\206 Neuronal survival. Neurotrophins promote the survival of spe- cifi c types of neu rons during development. NGF is present in the skin of the rat from birth (Constantinou et al., 1994), and is essential for nociceptor survival (Kirstein and Farinas, 2002). BDNF pro duced within sensory ganglia also plays a role in survival of nociceptive neurons responsiv e to NGF and GDNF (Valdes-Sanchez et al., 2010). \227\206 TRPA1 is activated by noxious cold (<18 C in some but not all studies), and by p ungent compounds such as horseradish, mustard, and formalin (Patapoutian et al., 2009). Res ponses to mustard oil have been recorded from DRG neurons in early development (Fitzgerald, 1987b, 1987c), but there is limited direct activation of spinal refl ex responses in the f i rst postnatal week (Fitzgerald and Gibson, 1984; Walker et al., 2007) when lev- els of TR PA1 mRNA are low in DRG neurons (Hjerling-Leffl er et al., 2007) and central C-fi bre conne ctions are immature. \227\206 Innervation. Th e pattern and density of skin innervation is regu- lated by access to local neurotrophic factors. \227\206 Nociceptor diff erentiation. Levels of NGF and neurotrophin-3 (NT3) regulate the diff erentiation of A high-threshold mech- anoreceptors and C-fi bre nociceptors. \227\206 TRPM8 is activated by innocuous cold and cooling compounds such as menthol (Wang and Woolf, 2005). TRPM8 mRNA is present in DRG neurons at levels similar to the adult at P0 , and although levels are reduced at P7 and P14, responses to menthol are present (Hjerling -Leffl er et al., 2007). \227\206 DRG neuronal phenotype. During embryonic development, 70% to 80% of DRG neurons a nd almost all that project to the superfi - cial dorsal horn express trkA and are NGF depen dent. A subpop- ulation of neurons downregulate expression of trkA around the time of birth , and begin to express Ret, the signaling component for GDNF, leading to a postnatal increa se in this IB4+ve popu- lation (Beland and Fitzgerald, 2001; Fitzgerald, 2005). Neurons tha t continue to express trkA remain as the peptidergic popula- tion and express CGRP and SP ( Guo et al., 2001). \227\206 Purinergic receptors comprise a P2X family of ionotropic channels and a P2Y famil y of G-protein coupled receptors. The P2X 3 receptor and P2X 2/3 heteromultimers have the h igh- est level of expression in sensory neurons. P2X 3 receptors on the sensory terminals o f IB4+ve subpopulation of nocicep- tive neurons respond to adenosine triphosphate (ATP) tha t has been released from endothelial and Merkel cells and from sympathetic neurons. P2X 7 r eceptors also play an important role in inflammatory hyperalgesia (Burnstock, 2006). In ear ly development, a higher proportion of DRG neurons express P2X 3 receptors, and this gradua lly becomes limited to IB4+ve neurons. In addition, P2X 3 agonists evoke a greater degree o f depolarization and more marked behavioral response. There is also a reciprocal relationsh ip with P2X 7 receptor expres- sion on satellite glial cells, which increases as P2X 3 rece p- tor expression decreases throughout postnatal development (Chen et al., 2012). Nociceptors and transduction In adulthood, nociceptors have high thresholds for activation and respond to stimuli of suffi cient intensity to cause damage. In addi- tion, repeated st imuli can result in sensitization of the peripheral terminal, and release of neuropeptides from the terminal contrib- utes to local vasodilation and erythema. DRG neurons respond to a range of peripheral mechanical, thermal, and chemical stimuli at birth (Fitzgerald, 1987b , 1987c). Age-related changes in receptor function and distribution, and in fi ring frequen cy, can alter sensi- tivity to diff erent stimuli, but nociceptors are responsive to noxiou s stimuli and respond to tissue damage aft er birth (Fitzgerald and Walker, 2009). SECTION 2 biological basis of pediatric pain 56 Mechanical stimuli Mechanoreceptors detect pressure stimuli at a range of intensities, and in vivo may be classifi ed according to their threshold. A range of additional mechanical s timuli (e.g. light brush, texture, vibra- tion, touch) can be detected via specialized nerv e endings in skin and in association with specialized structures such as hair follicles, Me issner corpuscles, or Pacinian corpuscles (Delmas et al., 2011). Glabrous skin in the dista l extremities (e.g. soles of feet/hindpaws) is more densely innervated than hairy skin, wit h a higher propor- tion of high threshold and faster conducting mechanosensitive fi bres (B oada et al., 2010). to increased sensitivity in the area surrounding the injured tissue, and is centrally-media ted. Although initially advantageous, ongoing sensitization of noci- ceptive responses can becom e maladaptive and contribute to per- sistent pain states (Hucho and Levine, 2007; Woolf and Ma, 2007). It has recently been suggested that nociceptive pathways may be described not j ust as being in a na ve or a sensitized state, but that prior injury may induce a \200 \230primed \200\231 state, in which the system is more responsive to future injury, such th at lower concentrations of mediators can elicit enhanced and prolonged hyperalgesia (Hucho and Levine, 2007). In early life, peripheral sensory fi bres are also responsive to Low threshold mechanoreceptors (LTMs) respond to innocu- ous levels of pressure and are gen erally associated with A nerve endings. High-threshold mechanoreceptors (HTMs) are activ ated by noxious or injurious mechanical forces, and are associated with A and polymodal C fi bres. Mechanosensitive neurons can also be diff erentiated by their fi ring frequency which encodes features of the mechanical stimulus (i.e. rapidly, intermediately, slowly, or ultra-slowly adapting), with nociceptive responses predominantly mediated by slowly and ul tra-slowly adapting currents (Delmas et al., 2011). Transduction is thought to be the resul t of an ion chan- nel that is opened directly by mechanical force, and candidate ion channe ls include acid-sensing ion channels (ASICs) and members of the TRP family (Woolf and Ma, 2 007). changing levels of activity in two diff erent manners: \227\206 Repeated stimuli can produce acute peripheral sensitization or hyperalgesia as seen at older ages (see later). \227\206 Activity is required for strengthening synaptic connections and pathway developme nt. During normal physiological develop- ment, activation of nociceptors is rare, and low t hreshold A-fi bre input contributes to shaping of nociceptive circuits (Waldenstrom et al., 2003). Altering this balance of activity during critical peri- ods in early life impairs n ormal development. Excessive reduc- tions in aff erent activity can alter synaptic function , structural and somatotopic organization in the spinal cord, and increase neuronal apoptos is resulting in long-term decreases in sensory thresholds (Beggs et al., 2002; Fitzgerald, 2005; Granmo et al., 2008; Walker et al., 2010). Excessive increases in activity as a resul t of pain and injury at specifi c developmental stages can also alter future sensitivity in a manner that is not seen following the same injury at older ages (Walker et al., 2009; fo r further details see Walker, Chapter 3, this volume). Sodium channels Voltage-gated sodium channels not only play an important role in transmissi on of the action potential along the axon, but diff erent subtypes can modulate sensitivity , and are expressed along axons as well as in the peripheral intra-epidermal nerve endings (Dib-Hajj et al., 2010; Persson et al., 2010). Voltage-gated sodium channels (Na v ) may be either sensitive or resistant to tetrodotoxin (desig- nated as TTX-S and TTX-R respectivel y). Th e TTX-S subtypes Na v 1.3 and 1.7 and TTX-R subtypes Na v 1.8 and 1.9 are expressed by nociceptors. Post-translational modifi cations enhance the excit- ability of TTX-R chann els and contribute to peripheral nocicep- tor sensitization, and alterations in expression of Na v channels may occur in persistent pain states in adulthood (Lai et al., 2004; Liu an d Wood, 2011). C- \201 bre stimuli Sensitization of peripheral aff erents following noxious or repeated s timuli can be demonstrated from early development, but the degree of activation by diff ere nt stimuli can vary (Fitzgerald, 1987c, 1991b; Koltzenburg and Lewin, 1997). Mustard oil, w hich acts on TRPA1 receptors, produces only weak aff erent responses in neonatal rat DRG re cordings (Fitzgerald, 1987c), but does sensitize the nocic- eptor to subsequent stimuli wit hin the area of injury, i.e. primary hyperalgesia (Fitzgerald, 1991b), although to a lesser degree than at older ages (P3 < P10 < P21; Walker et al., 2007). By contrast, sec- ondary hyperalgesia, which is dependent on central mechanisms, is not induced by C-fi bre stimuli (capsaicin or mustard oil) in the fi rst postnatal week, but has reached adult levels by th e third postnatal week (i.e. approximately late childhood; Jiang and Gebhart, 1998; Walker et al., 2007). Both TTX-S and TTX-R Na channels can be identifi ed from birth in rat DRG (Ogata and Tateba yashi, 1992). Na v 1.3 is expressed at higher levels during embryonic development. Th e TTX -R sodium channels Na v 1.8 and Na v 1.9 are expressed on C-fi bres at birth and reach adul t levels by P7 (Benn et al., 2001). Peripheral sensitization Th e response to a given nociceptive stimulus can change with time , and sensitization of nociceptive pathways provides further warning and encourages protect ion of the injured part (Woolf and Ma, 2007). Hyperalgesia refers to an increased response to a noxious stimulus (increased response to suprathreshold stimuli) and allodynia refers t o pain or nociceptor fi ring in response to a previously non-noxious stimulus (reduction in threshold and spontaneous activity). Primary hyperalgesia or peripheral sen- sitization is the result of altered nociceptor sensitivity, with an increased response to thermal and me chanical stimuli within the area of tissue damage. By contrast, secondary hyperalgesia refe rs In \202 ammation Peripheral infl ammation results in pain, heat, and redness (due to vasod ilation), and swelling (due to plasma extravasation). An \200\230infl ammatory soup \200 \231 that is \200\230tasted \200\231 by receptors on nerve terminals (Woolf and Ma, 2007) is made up of multiple mediators includ- ing: prostaglandins (Kawabata, 2011), leukotrienes (Noguchi and Okubo, 2011), substance P, nitric oxide, cytokines, chemokines, bradykinin, s erotonin, histamine, tumor necrosis factor (TNF ) and neurotrophic factors (e.g. NGF). F ollowing release from the nerve terminal, or from infl ammatory cells or damaged tissue, CHAPTER 6 nociceptive signaling in the periphery and spinal cord 57 these mediators activate and/or sensitize the nociceptor terminal by acting on specifi c se nsory receptors that include (Woolf and Ma, 2007; Linley et al., 2010): et al., 1997; Suzuki et al., 2002). Importantly, the functional prop- erties of the SDH net work are highly age-dependent. As a result, understanding how the immature SDH is organized at a cellular and molecular level may yield new insight into novel approaches to regulate the ascending fl ow of noxious information to the brain, and resultant pain perception, in infants and children. \227\206 G protein-coupled receptors (e.g. bradykinin B1 and B2; pro- tease-activated receptors, PAR 1 \200\2233; histamine receptors). \227\206 Receptor tyrosine kinases (e.g. growth factor receptors such as trkA). Electrophysiological properties of neonatal dorsal horn neurons Extracellular single-unit r ecordings in intact, anesthetized rodents clearly demonstrated that immature dorsal horn ne urons are char- acterized by low thresholds, large peripheral receptive fi elds, and prolon ged action potential aft er-discharges compared to adult dorsal horn cells (Fitzgerald, 198 5; Torsney and Fitzgerald, 2002). Repetitive low-threshold stimulation also leads to a prog ressive sensitization of neonatal dorsal horn neurons, which disappears by P21 (Jennings an d Fitzgerald, 1998). Notably, these same functional properties also characterize nociceptiv e withdrawal refl exes in the newborn rodent (Schouenborg, 2003), kitten (Ekholm, 1967), an d human (Andrews and Fitzgerald, 1994; Andrews et al., 2002). Th e developmental alteration s in the response of dorsal horn neurons following stimulation of their peripheral receptiv e fi elds may be explained by three factors: \227\206 Ionotropic receptors/ion channels, such as TRP channels (Patapoutian et al., 2009 ), purinergic receptors (Burnstock, 2006), and acid-sensitive channels. Infl ammatory mediators initiate intracellular signaling cas- cades, involving activation o f a range of protein kinases (Bhave and Gereau, 2004; Hucho and Levine, 2007) and mediate i ncreased sen- sitivity by: \227\206 Rapid response post-translational changes which directly modu- late ion channel a ctivity, change membrane potential, or increase insertion of receptors into the membrane (B have and Gereau, 2004). \227\206 Alterations in transcriptional programs and changes in gene expression resulting in longer-term altered sensitivity (Linley et al., 2010), such as changes in the expression and transportation of pro-nociceptive molecules (e.g. substance P, NGF) and ion channels. \227\206 Developmental changes in sensory transduction and/or the func- tional properties of primary aff erent neurons. Infl ammatory hyperalgesia occurs from early development. Responses to thermal stimuli are enhanced following applica- tion of infl ammatory mediators in skin-nerve preparations from embryonic chick (Koltzenburg and Lewin, 1997). Infl ammatory stimuli, such as carrageenan and complete Freund \200\231s adjuvant (CFA), produce hyperalgesia in neonatal rat pups, an d the degree and duration of response varies with the dose and type of agent (Ren et al., 2 004; Walker et al., 2003). Hindpaw infl ammation also accelerates the postnatal upregulatio n of IB4+ve neurons in the DRG (Beland and Fitzgerald, 2001). Although the pattern of respo nse changes with age, hindpaw formalin injection also pro- duces a signifi cant behavioral response in rat pups (Guy and Abbott, 1992). \227\206 Age-dependent changes in the intrinsic membrane proper- ties of dorsal horn cells which infl uence the level of neuronal excitability. \227\206 Th e maturation of excitatory and inhibitory synaptic inputs onto dorsal horn neu rons. Th is section of the chapter will focus on the last two factors. Intrinsic excitability of developing SDH neurons An important feature of developing CNS net works is spontaneous activity (SA), which can be generated by a variety of mechanisms (Blan kenship and Feller, 2010) and contributes to both early cir- cuit formation (Tritsch et al. , 2007) and the subsequent establish- ment of appropriate sensory maps (Kandler et al., 200 9). SA in the newborn SDH is predominantly localized to lamina I, and is then downregulated with age (J. Li and Baccei, 2011a). During early life, a subset of glutamatergic interneur ons within lamina I func- tion as \200\230pacemaker \200\231 neurons that are capable of g enerating oscil- latory burst-fi ring. Th is is independent of synaptic transmission within the network and instead relies on a combination of intrin- sic voltage-gated conductances (including persistent Na + currents and high-threshold N-type and L-type Ca 2+ channels). A s these pacemaker neurons are innervated by nociceptive aff erents and project throughout t he dorsal \200\223ventral axis of the immature spinal cord, they may provide an endogenous source of activity to the developing sensorimotor circuits mediating spinal nociceptive ref l exes (J. Li and Baccei, 2011a). Alternatively, they may serve to synchronize the fi ring of ascending spinal projection neurons in the neonate. In this context, burst-fi ring also characterizes soma- tosensory processing in the developing human brain (Fabrizi et al., 201 1). Th e acute and potential long-term eff ects of neonatal injury in diff erent laboratory mod els are further discussed by Walker (Chapter 3, this volume). Nociceptive processing in the spinal cord Th e processing of noxious information by the CNS begins in the superfi cial dorsal horn (SDH) of the spinal cord (laminae I \200\223II), wh ich receives direct projections from nociceptive primary aff er- ent neurons (A and C fi bres) with cell bodies in the DRG. Th e vast majority (>95%) of neurons within the SDH cor respond to propriospinal and local circuit interneurons (Bice and Beal, 1997a, 1997b). As a result, the SDH network has the ability to integrate sensory inputs, which vary in modalit y, intensity, and location, and transform them into an output signal, which ascends to the brain. Th is output of the SDH circuit is conveyed by a small percentage of lamina I neuron s ( c .5%) which project to multiple supraspinal sites (Spike et al., 2003) and are known t o be instrumental for the gen- eration of chronic pain following tissue or nerve injury (Ma ntyh SECTION 2 biological basis of pediatric pain 58