Brun Sundblad, G. M., Saartok, T., and Engstr m, L. M. T. (2007). Prevalence and co-occu rrence of self-rated pain and perceived health in school- children: age and gender diff ere nces. Eur J Pain, 11 (2), 171 \200\223180 . Bugdayci, R., Ozge, A., Sasmaz, T., Oner Kurt, A., Kaleagasi, H., Karakelle, A., et al . (2 005). Prevalence and factors aff ecting headache in Turkish schoolchildren. Pediatr Int, 47 (3), 316 \200\223322 . When he was 3 years old, Jackson woke several times in the middle of the night with pain in his knees. He would cry out for his parents, they would give him ibuprofen, massage his ba ck, or, on occasion, put him in a warm bath. By his fi ft h birthday these episodes had res olved. Buskila, D., Neumann, L., Zmora, E., Feldman, M., Bolotin, A., and Press, J. (2003). Pain s ensitivity in prematurely born adolescents. Arch Pediatr Adolesc Med , 157 (11), 1079 \200 \22382 . Prior to starting school Jackson had developed a fear of receiv- ing his immunizations \200 \224a problem which persists to the present day. For his yearly fl u vaccination Jackson re ceives lidocaine prilocaine cream, and learned some guided imagery techniques from a family friend. His fear of needles extended to his interac- tions with his dentist and he require d general anesthesia to have three cavities fi lled. Campo, J. V., Di Lorenzo, C., Chiappetta, L., Bridge, J., Colborn, D. K., Gartner, J. C., e t al . (2001). Adult outcomes of pediatric recurrent abdominal pain: do they just grow out of it? Pediatrics , 108 (1), E1 . Carbajal, R., Rousset, A., Danan, C., Coquery, S., Nolent, P., Ducrocq, S., et al . (2008). Epidemiology and treatment of painful procedures in neonates in intensive care units. JAMA , 300 (1), 60 \200\22370 . Chitkara, D. K., Talley, N. J., Weaver, A. L., Katusic, S. K., De Schepper, H., Rucker, M. J., et al . (2007). Incidence of presentation of common functional gastrointestinal disorde rs in children from birth to 5 years: a cohort study. Clin Gastroenterol Hepatol, 5 (2), 18 6 \200\223191 . At age 11 Jackson collided with a teammate while playing soc- cer. He landed on his back. H e complained about pain in his lower back for several weeks before his parents sought medic al treatment. He was diagnosed with a muscular strain and received physical therapy for 2 m onths before he could return to full activ- ity without pain. Clancy, C. A., McGrath, P. J., and Oddson, B. E. (2005). Pain in children and adolescents with spina bifi da. Dev Med Child Neurol , 47 , 481 \200\22391 . Cummings, E. A., Reid, G. J., Finley, G. A., McGrath, P. J., and Ritchie, J. A. (1996). Pre valence and source of pain in pediatric inpatients. Pain , 68 (1), 25 \200\22331 . SECTION 1 introduction 18 Johnston, C. C., Collinge, J. M., Henderson, S., and Anand, K. J. (1997). A cross sectional survey of pain and analgesia in Canadian neonatal intensive care units. Clin J Pain , 3 (4 ), 1 \200\2235 . Dampier, C., Ely, B., Brodecki, D., and O \200\231Neal, P. (2002a). Characteristics of pain managed at home in children and adolescents with sickle cell disease by using diary self-r eports. J Pain, 3 (6), 461 \200\223470 . Johnston, C. C., Barrington, K. J., Taddio, A., Cabajal, R., and Filion, F. (2011). Pain in Canadian NICUs. Have we improved over the past 12 years? Clin J Pain, 27 (3), 225 \200\223 232 . Dampier, C., Ely, E., Brodecki, D., and O \200\231Neal, P. (2002b). Home management of pain in sickle cell disease: a daily diary study in children and adolescents. J Pediatr Hematol Oncol, 24 (8), 643 \200\223647 . Kashikar-Zuck, S., Goldschneider K. R., Powers, S. W., Vaught, M. H., and Hershey, A. D. (2 001). Depression and functional disability in chronic pediatric pain. Clin J Pain , 17 (4), 341 \200\2239 . Dangel, T. (2005). 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Pain in long-term adult survivors of childhood cancers and their siblings: a report fro m the Childhood Cancer Survivor Study. Pain, 152 (11), 2616 \200\2232624 . Engel, J. M., Petrina, T. J., Dugeon, B. J., and McKearnan, K. A. (2005). Cerebral palsy an d chronic pain: a descriptive study of children and adolescents. Phys Occup Th er Pediatr, 25 (4), 73 \200\22384 . Malleson, P. N., Oen, K., Cabral, D. A., Petty, R. E., Rosenberg, A. M., and Cheang, M. (20 04). Predictors of pain in children with established juvenile rheumatoid arthritis. Arthrit is Rheum, 51 (2), 222 \200\223227 . Evans, A. M. and Scutter, S. D. (2004). Prevalence of \200\230growing pains \200\231 in yo ung children. J Pediatr, 145 (2), 255 \200\2238 . Fernandez, C. V. and Rees, E. P. (1994). Pain management in Canadian level Masiero, S., Carraro, E., Sarto, D., Bonaldo, L., and Ferraro, C. (2010). Healthcare servic e use in adolescents with non-specifi c musculoskeletal pain. Acta P diatr, 99 , 1224 \200\2231228 . 3 neonatal intensive care units. CMAJ , 150 (4), 469 \200\223470 . Ghandour, R. M., Overpeck, M. D., Huang, Z. J., Kogan, M. D., and Scheidt, P. C. (2004). He adache, stomachache, backache, and morning fatigue among adolescent girls in the United Sta tes: associations with behavioral, sociodemographic, and environmental factors. Arch Pediat r Adolesc Med, 158 (8), 797 \200\223803 . Mikkelsson, M., El-Metwally, A., Kautiainen, H., Auvinen, A., Macfarlane, G. J., and Salmin en, J. J. (2008). Onset, prognosis and risk factors for widespread pain in schoolchildren: a prospective 4-year follow-up study. Pain, 138 , 681 \200\223687 . Goldman, A., Hewitt, M., Collins, G. S., Childs, M., and Hain, R. (2006). Symptoms in child ren/young people with progressive malignant disease: United Kingdom Children \200\231s Canc er Study Group/Pediatric Oncology Nurses Forum survey. Pediatrics, 117(6), e1179 \200\223e1 186 . Mikkelsson, M., Salminen, J. J., and Kautiainen, H. (1997). Non-specifi c musculoskeletal p ain in preadolescents. Prevalence and 1-year persistence. Pain, 73 (1), 29 \200\22335 . Moura-Leite, F. R., Ramos-Jorge, M. L., Bonanato, K., Paiva, S. M., Vale, M. P., and Pordeu s, I. A. (2008). Prevalence, intensity and impact of dental pain in 5-year-old preschool ch ildren. Oral Health Prev Dent, 6 (4), 295 \200\223301 . Murray, L. J., McCarron, P., Boreha m, C. A., McGartland, C. P., and Johnston, B. T. (2007). Prevalence of epigastric pain, hea rtburn and acid regurgitation in adolescents and their parents: evidence for intergeneratio nal association. Eur J Gastroenterol Hepatol, 19 (4), 297 \200\223303 . Goodman, J. E. and McGrath, P. J. (1991). Th e epidemiology of pain in children and adolescents: a review. Pain , 46 (3), 247 \200\223264 . Groenewald, C. B., Rabbitts, J. A., Schroeder, D. R., and Harrison, T. E. (2012). Prevalenc e of moderate-severe pain in hospitalized children. Pediatr Anaesth , 22 (7), 661 \200\2236 68 . Hamilton, A. B. and Zeltzer, L. K. (1994). Visceral pain in infants. J Pediatr, 125 (6II), S95 \200\223S102 . Parkinson, K. N., Gibson, L., Dickinson, H. O., and Colver, A.F. (2009). Pain in children w ith cerebral palsy: a cross-sectional multicentre European study. Acta Paediatr, 99 (3), 44 6 \200\223451 . Headache Classifi cation Subcommittee of the International Headache Society (2004). Th e In ternational Classifi cation of Headache Disorders: 2nd edition. Cephalalgia, 24 (S1), 9 \200\223160 . Perquin, C. W., Hazebroek-Kampschreur, A. A., Hunfeld, J. A., Bohnen, A. M., Van Suijlekom- Smit, L. W., Passchier J., et al . (2000). Pain in children and adolescents: a common exper ience. Pain, 87 (1), 51 \200\22358 . Houlihan, C., O \200\231Donnell, M., Conaway, M., and Stevenson, R. D. (2004). Bodily pain and health-related quality of life in children with cerebral palsy. Dev Med Child Neurol, 4 6 , 305 \200\223310 . Petersen, S., Bergstrom, E., and Brulin, C. (2003). High prevalence of tiredness and pain i n young schoolchildren. Scand J Public Health, 31 , 367 \200\223374 . Pillai Riddell, R., S tevens, B., McKeever, P., Gibbins, S., Asztalos, E., Katz, J., et al . (2009). Chronic pain in hospitalized infants: health professionals \200\231 perspectives. J Pain, 10 (12), 1217 \200\2231225 . Huguet, A. and Miro, J. (2008). Th e severity of chronic pediatric pain: an epidemiological study. J Pain, 9 (3), 226 \200\223236 . Hyams, J., Burke, G., Davis, P., Rzepski, B., and Andrulonis, P. A. (1996). Abdominal pain and irritable bowel syndrome in adolescents: a community-based study. J Pediatr, 129 (2), 2 20 \200\223226 . Platt, O. S., Th orington, B. D., Brambilla, D. J., Milner, P. F., Rosse, W. F., Vichinsky, E., et al . (1991). Pain in sickle cell disease. Rates and risk factors. N Engl J Med, 325 (1), 11 \200\22316 . Jacob, E., Hesselgrave, J., Sambuco, G., and Hockenberry, M. (2007). Variations in pain, sl eep, and activity during hospitalization in children with cancer. J Pediatr Oncol Nurs, 24 (4), 208 \200\223219 . CHAPTER 2 prevalence and distribution of pain in children 19 Stevens, B. J., Abbott, L. K., Yamada, J. Harrison, D., Stinson, J., Taddio, A., et al . (2 011). Epidemiology and management of painful procedures in children in Canadian hospitals. CMAJ , 183 (7), e403 \200\223e410 . Porter, F. L., Wolf, C. M., Gold, J., Lotsoff , D., and Miller, J.P. (1997). Pain and pain management in newborn infants: a survey of physicians and nurses. Pediatrics , 100 (104), 6 26 \200\223632 . Taddio, A. (1999). Eff ects of early pain experience: the human literature. In P. J. McGrat h and G. A. Finley (eds) Chronic and Recurrent Pain in Children and Adolescents , pp. 57 \200\22374. Seattle : IASP Press . Ramchandani, P. G., Hotopf, M., Sandhu, B., and Stein, A. (2005). Th e epidemiology of recu rrent abdominal pain from 2 to 6 years of age: results of a large, population-based study. Pediatrics, 116 (1), 46 \200\22350 . Ramstad, K., Jahnsen, R., Skjeldal, O. H., and Diseth, T. H. (2011). Characteristics of recurrent musculoskeletal pain in children with cerebral palsy aged 8 to 18 years. Dev Med Child Neurol, 53 , 1013 \200\2231018 . Taddio, A., Shah, V., Gilbert-Macleod, C., and Katz, J. (2002). Conditioning and hyperalges ia in newborns exposed to repeated heel lances. JAMA , 288 , 857 \200\22361 . Taddio, A., Ipp, M., Thivakaran, S., Jamal, A., Parikh, C., Smart, S., et al . (2012). Surv ey of the prevalence of immunization non-compliance due to needle fears in children and adu lts. Vaccine, 30 , 4807 \200\2234812 . Taylor, E. M., Boyer, K., and Campbell, F. A. (2008) . Pain in hospitalized children: a prospective cross-sectional survey of pain prevalence, i ntensity, assessment and management in a Canadian pediatric teaching hospital. Pain Res Man ag , 13 , 25 \200\22332 . Ratnayake, N. and Ekanayake, L. (2005). Prevalence and impact of oral pain in 8-year-old ch ildren in Sri Lanka. Int J Paediatr Dent, 15 (2), 105 \200\223112 . Schanberg, L. E., Lefebvre, J. C., Keefe, F. J., Kredich, D. W., and Gil, K. M. (1997). Pai n coping and the pain experience in children with juvenile chronic arthritis. Pain, 73 (2), 181 \200\223189 . Th ompson, K. L., Varni, J. W., and Hanson, V. (1987). Comprehensive assessment of pain in juvenile rheumatoid arthritis: an empirical model. J Pediatr Psychol, 12 (2), 241 \200\2232 55 . Schanberg, L. E., Anthony, K. K., Gil, K. M. and Maurin, E. C. (2003). Daily pain and sympt oms in children with polyarticular arthritis. Arthritis Rheum, 48 (5), 1390 \200\2231397 . T z n, E. H., Guven, D. K., and Eker, L. (2010). Pain prevalence and its impact on th e quality of life in a sample of Turkish children with cerebral palsy. Disabil Rehabil, 32 (9), 723 \200\223728 . Simons, S. H., Van Dijk, M., Anand, K. J. S. Rooft hooft , D., Van Lingen, R., and Tibboel, D. (2003). Do we still hurt newborn babies? A prospective study of procedural pain and ana lgesia in neonates. Arch Pediatri Adolesc Med, 157 (11), 1058 \200\2231064 . Vandvik, I. H. and Eckblad, G. (1990). Relationship between pain, disease severity and psyc hosocial function in patients with juvenile chronic arthritis (JCA ). Scand J Rheumatol, 19 (4), 295 \200\223302 . Slade, G. D. (2001). Epidemiology of dental pain and dental caries among children and adolescents. Community Dent Health, 18 , 219 \200\223227 . Van Dijk, M. A., McGrath, P.A., Pickett, W., and Van Den Kerkhof, E. G. (2006). Pain preval ence in nine- to 13-year-old school children. Pain Res Manag, 11 (4), 234 \200\223240 . Stallard, P., Williams, L., Lenton, S., and Vellemn, R. (2001). Pain in cognitively impaire d, non-communicating children. Arch Dis Child, 85 (6), 460 \200\223462 . Stanford, E. A., Chambers, C. T., Biesanz, J. C., and Chen, E. (2008). Th e frequency, traj ectories and predictors of adolescent recurrent pain: a population-based approach. Pain, 13 8 , 11 \200\22321 . Wilson, A. C., Samuelson, B., and Palermo, T. M. (2010). Obesity in children and adolescent s with chronic pain: associations with pain and activity limitations. Clin J Pain, 26 (8), 705 \200\223711 . Stevens, B., McGrath, P., Gibbins, S., Beyene, J., Breau, L., Camfi eld, C., et al . (2003) . Procedural pain in newborns at risk for neurologic impairment. Pain , 105 ( 1 \200\2232 ) , 27 \200\22335 . Zapata, A. L., Moraes, A. J. P., Leone, C., Doria-Filho, U., and Silva, C. A. A. (2006). Pa in and musculoskeletal pain syndromes in adolescents. J Adolesc Health, 38 (6), 769 \200 \223771 . CHAPTER 3 Long-term e \200 ects of early pain and injury: animal models Suellen M. Walker Summary Nociceptive pathways are functional following birth and acute responses to noxious stimuli have been documented from early development in both clinical and laboratory studies . Th e ability of noxious aff erent input to alter the level of sensitivity of nociceptive pathways in the adult nervous system, with, for example the devel- opment of central sensit ization, is well established (Woolf, 2011). However, the developing nervous system has addi tional suscepti- bilities to alterations in neural activity, and increases due to pain and injury in early life may produce eff ects not seen following the same input at older ages. As a result, early tissue injury may lead to persistent changes in somatosensory processing and altered sensi- tivity to future noxious stimuli. Th e impact of early pain and injury cannot be simply viewed as increasing or decreasing sensitivity as results vary depending o n the type and severity of injury and the outcomes used for assessment. Laboratory studies allow evaluation of diff erent forms of injury, potential confounding factors, underly- ing mechanisms, and potential for modulation by analgesia. Th is chapter will fi rstly summarize factors that infl uence long- term eff ects in animal models with examples from diff erent stud- ies, and secondly, describe in more detail the specifi c eff ects of diff erent forms of injury. In line with clinical reports, the majori ty of studies focus on injuries sustained within the neonatal period and early life. Labora tory studies allow evaluation of neural proc- esses underlying the encoding and processing of noxious stimuli (i.e. \200\230nociception \200\231), whereas the term \200\230pain \200\231 may be reserved for the human experience which encompasses cognitive and emotional dimensions; but for the purposes of this chapter \200\230pain \200\231 will also be used in association with behavioral and tissue responses to injury in animals. Evaluating the long-term impact of early pain: general principles A number of diff erent va riables and outcomes can be assessed in preclinical studies evaluating the long-term impact of early pain, and as seen with clinical evidence, studies vary in their design and method ology. Introduction Persistent alterations in pain sensitivity following early pain experi- ence i n neonates and infants are outlined by Grunau (Chapter 4, this volume). Clinical evaluation s vary in their methodology, and it is clear that early pain experience does not simply eit her increase or decrease pain sensitivity. Preclinical studies similarly document dif- feri ng long-term consequences of early pain and injury (Fitzgerald and Walker, 2009), and allow further evaluation of: Extrapolation across species Understanding the potential clinical signifi cance of early li fe injury in animal models requires extrapolation to a similar developmen- tal stage in hum ans. While direct translation of developmental age from rodents to humans, and the specifi c timing of events aft er birth, continues to be debated, the sequence of development of se nsory and refl ex systems in rodents correlate with those of human infants (Wood et al., 20 03). Statistical models have been developed to translate development across species (Clancy et al., 2007b; Nagarajan et al., 2010), but are predominantly based on structural measures , and acknowledge that they cannot account for activity-dependent modifi cation following b irth (Clancy et al., 2007a). Maturation varies in diff erent regions of the peripheral and central nervous systems, but in terms of spinal processing many approximate a postnatal day (P)3 rat with a preterm human neonate, P7 with an infant, P21 with an adolescent, and P35 with \231 Th e impact of diff erent types and severity of early injury. \231 Responses at diff erent developmental stages to determine if there are critical peri ods during which alterations in normal develop- ment produce long-term eff ects. \231 Th e mechanisms underlying persistent sensory changes. \231 Th e degree to which the impact of early injury can be modulated by analgesia. CHAPTER 3 long-term effects of early pain and injury: animal models 21 young adulthood (Brambrink et al., 2010; Fitzgerald and Walker, 2009; McCutcheon and Marine lli, 2009; see also Walker and Baccei, Chapter 6, and Fitzgerald, Chapter 8, this volume fo r further details of the developmental changes in nociceptive signaling). (e.g. hindpaw or gut), directly on the spinal cord, or by injection in other sites within t he central nervous system (CNS). \231 Detailed evaluation of activity in diff erent ion channels at a cellu- lar level. Pa tch clamp techniques on neurons within nociceptive pathways may be performed in slices of C NS tissue, including spinal cord, or in vivo . An additional diffi culty relates to evaluating the potential \200\230eff ect size \200 \231 of observations in animals models. For example, what degree of change in baseline sens ory thresholds is likely to correlate with a clinically signifi cant eff ect? Enhanced resp onses to future pain, which can result in alterations in the degree and duration of hyper- algesia, are more quantifi able and perhaps easier to translate to the clinical setting. Responses to stimuli of varying intensity can reveal diff erences in the distribution, time course, and underlying mechanisms of altered sensory processing. For example, neonatal inf l ammation produces late-onset generalized baseline hypoalgesia, as noted ear- lier in this section. In addition, an enhanced segmental hyperal- gesia is unmasked by subsequent infl ammation or incision in the previously injured, but not the contralateral paw (Chu et al., 2007; Ren et al., 2004). Ideally, eff ects in several species would be evaluated, and although primate studies are b eing performed to evaluate potential adverse eff ects of general anesthesia in early develo pment (Brambrink et al., 2010), resources for primate studies are limited. Th e majority of pain studies are performed in rats or mice, and the rodent has several advantages as a dev elopmental model: Th e degree of hyperalgesia following injury in later life is fre- quently assessed. Th is may be in response to the same form of injury, such as repeat surgical incision (Beggs et a l., 2012b) or re-infl ammation of the hindpaw (Ren et al., 2004). Alternatively, diff erent stimuli may be used to assess specifi c mechanisms (e.g. evaluation of C-fi bre mediated r esponses by subsequent injection of capsaicin; Hohmann et al., 2005) or to evaluate the gen eraliz- ability of altered sensitivity to diff erent forms of injury (e.g. initial somatic injury and subsequent visceral sensitivity). \231 Rodents are born at a relatively immature stage, thus allow- ing evaluation of eff e cts that correlate with the preterm human infant. Mice and rats have similar gestation peri ods (19 and 21 days respectively) and then mature at similar rates. \231 Rat pups are weaned at P21 and reach adulthood by 6 to 8 weeks of age, so persistent eff ects can be followed across a relatively compressed lifespan. Age at time of injury Th e majority of current clinical and laboratory research focuses on long-term eff ects following pain experienced in the neonatal period and/or early infancy. An important feature of injury is early life is that eff ects in the developing nervous sys tem may diff er from those seen following the same injury at older ages. Th ere are \200 \230critical peri- ods \200\231 when changing levels of neural activity can alter neural de vel- opment (Hensch, 2004), but this period of vulnerability diff ers in diff erent regions of the nervous system, and is also dependent on the type of injury. \231 Control animals in the same litter can be used to assess potential confounding eff e cts due to handling and maternal separation. \231 Th e ability of analgesics to modify persistent eff ects can be stud- ied in a standardized comparative manner. Type of injury A range of injuries can be utilized to model the many diff erent sources of pain that may be experienced in early life, including: surgical injury, infl ammation, repe ated needle sticks, and visceral insults (see later sections for specifi c details). Th is allows evaluation of the response to specifi c components of various injuries, under- lying mechanisms, and also allows comparison of the diff erential sensitivity to analgesic inter ventions. To confi rm specifi c developmental or age-dependent responses, the same injury must be per formed and compared across a number of postnatal ages. In addition, the same intensity of s timulus should be delivered at each age. For example, the volume of infl ammatory agent or irritant solution injected at diff erent ages needs to be standardized for the rapidly chan ging size and weight of the animal, to confi rm that enhanced eff ects following early inju ry are not due to a more severe injury or more intense acute stimulus in the youngest anima ls. Th e volume of the C-fi bre stimulant mustard oil applied to the hindpaw has been adjus ted according to the size or \200\230volume \200\231 of the paw at dif- ferent ages (Jiang and Gebhart, 1998), the volume of capsaicin altered to produce comparative local responses (S. M. Walker et al., 2007), and anatomical landmarks used to produce incision of the same relative proportion of hindpaw at diff erent ages (S. M. Walker et al., 2009). Impact on sensory function and/or response to future injury Changes in baseline sensory sen sitivity or in the response to a subse- quent noxious stimulus in adulthood are the primary outcomes for assessing the long-term impact of prior pain and injury. Evaluation methods m ay include, but are not limited to, the following: \231 Changes in behavioral sensory threshold, such as the threshold for hindlimb withdraw al from a mechanical stimulus applied to the hindpaw or the latency to withdraw from a ther mal stimulus. A decrease in the mechanical threshold or thermal latency for withdrawal indi cates increased sensitivity or hyper algesia, and conversely an increase in threshold or la tency indicates decreased sensitivity or hypo algesia. Time interval before evaluation Th e impact of neonatal injuries on diff erent outcomes may change with time. Ideally the time course of response should be evaluated by regular asses sments until the animal or the system being evaluated is fully mature, as rodents may be co nsidered \200\230adult \200\231 at diff erent ages (McCutcheon and Marinelli, 2009). Perip heral nociceptive mechanisms mature relatively early, but spinal cord \231 In vivo electrophysiological recordings. Electromyography (EMG) can further quantify refl ex sensitivity to threshold and suprathreshold stimuli. Extracellular recordings from cells in the dorsal horn assess responses to physical, chemical, or electri- cal stimuli o r drugs; these may be applied to peripheral tissue SECTION 1 introduction 22 processing, and particularly modulation by descending pathways from the brainstem (Hathway et al., 2009, 2012) is not mature until the 3rd to 4th postnatal week (see Fitzgerald, Chap ter 8, this volume). Following neonatal infl ammation, a clear change is seen as the animal matures, with generalized hypoalgesia or decreased baseline sensitivity emerging only aft er P30 (Ren et al., 2004), potentially due to alterations in descending modulation (Zhang e t al., 2010). \231 Assessing the localized response to injury in the peripheral tissue (e.g. degree of persistent infl ammation or changes in innervation). \231 Evaluating changes in receptor expression in the cell bodies of peripheral aff erent s in the dorsal root ganglion (DRG) or spi- nal cord or other CNS regions using antibodies to the receptor (immunohistochemistry) or measuring receptor protein levels (Western blot). \231 Alterations in gene expression which indicate neuronal activa- tion (e.g. c-fos) or which relate to specifi c mechanisms mediating long-term consequences (e.g. upregulation or downregulation of genes controlling receptors, transmitters, or signaling pathways involve d in nociceptive function). Use of genetically modifi ed mice can also provide further insi ghts by comparison of wild- type strains with animals in which a portion of the gene is per - manently non-functional (knockout), inactivated in a tissue- or time-specifi c manner (co nditional knockout), or briefl y silenced (knockdown). Non-sensory behavioral outcomes Th e eff ect of early injury on anxiety in adulthood has be en assessed using established paradigms, but results vary with the type and inten- sity of initial stimulus. A low anxiety trait in adulthood (P50 \200\22355) followed early infl amm ation (0.25% carrageenan at P3) as assessed by: more time spent in the open arms of an elev ated plus maze; enhanced ability to cope with stress related to a forced swim test; and red uced basal and stress-related neuroendocrine markers at rest and following the swim test (A nseloni et al., 2005). Conversely, more severe hindpaw infl ammation (10 mcl of complete Fr eund \200\231s adjuvant (CFA) in P1 mice) increased anxiety behaviors in adult- hood (fewer entries into open arms of elevated plus maze at P90; Roizenblatt et al., 2010). Formalin i n P1 rats (10 mcl 4% into right forepaw and hindpaw) reduced exploratory behavior in males and increased anxiety (reduced time in open arm of elevated plus maze) in females (Negrigo et al., 2011). Similarly, daily formalin injections (5 mcl of 4%) into each hindpaw from P1 to P4 reduced exploratory behavior and impaired learning in a radial arm maze test (Anand et al., 2007; Rovnaghi et al., 2008). Repeated hindpaw needle prick- ing from P0 to P7 was also postulated to increase vulnerability to stress and anxiety-mediated behaviors, as defi cits in exploratory and defensive withdrawal behavior were found at P65, but without diff erences in stress hormones (Anand et al., 1999). Pharmacological interventions Pharmacological strategies may be used to further defi ne mec ha- nisms associated with the eff ects of early pain and injury. For exam- ple, in addition to immunohistochemistry studies showing altered microglial morphology and increased reacti vity, a reduction in hyperalgesia by administration of an inhibitor of microglia (e.g. mino cycline) further established the role of microglia in mediat- ing eff ects of prior neonata l incision (Beggs et al., 2012b). Similarly, prevention of long-term consequences by local anesthetic block- ade of peripheral nerves supplying the area of injury (S. M. Walker et al ., 2009), not only confi rms that activity within primary aff erent fi bres has a mechanist ic role, but also suggests a potential therapeu- tic intervention. Administration of drugs by diff erent routes can more closely evaluate the site of action: comparing responses fol- lowing intrathecal administration rather than systemic (oral, sub- cutaneous, intraperiton eal, intravenous) administration identifi es selective spinal cord eff ects; and localized small volume injections into the brain can evaluate the impact of altering function in diff er- ent brain regions. An early visceral insult (repeated colorectal distension from P8 to P21) also reduced explo ratory activity in adulthood (J. Wang et al., 2008). Interestingly, most models associated with increased anxiety also produced long-term increased pain sensitivity (reduced hind- li mb withdrawal sensory thresholds); whereas carrageenan infl am- mation produces generalized hypo algesia and reduced anxiety. Th is potential link between the pattern of pain and anx iety outcomes requires further evaluation. Assessing modulation of long-term eff ects by analgesics has important clinical implication s. Two diff erent aspects may be evaluated: (1) the ability of diff erent forms of analgesi a at the time of the initial insult to modify or prevent long-term conse- quences; and/or ( 2) the ability of diff erent analgesics to eff ectively manage any alterations in pain resp onse at the time of injury in later life. As central pathways involved in pain and reward processing are linked, the long-term impact of repeated neonatal hindpaw incision (at P3, P10, and P17) on motivational behavior in ad ulthood was tested. Exploratory behaviors in adulthood were not altered by neonatal incisio ns or anesthesia. Following a subsequent incision in adulthood, motivational behaviors were similar, but activity in orexin neurons in the lateral hypothalamus (an important region f or reward signaling) was increased (Low and Fitzgerald, 2012). Sex di \200 erences Th e degree to which male or female sex infl uences the susceptibility to long-term consequences of early injury varies, both with the type of initial injury and with the outcome being assessed. Although not all studies include comparison of female and male animals, altera- tions in either the degree or type of long-term impact of neona- tal interventions have been reported in some, but not all, studies and warrant further evaluat ion (Cataldo et al., 2010; LaPrairie and Murphy, 2007, 2010; Negrigo et al., 2011). At this stage, it is not possible to reliably conclude whether males or females are more susceptib le. For some behaviors the link with pain signaling is less clear. For example, ethanol prefere nce was reduced aft er repeat formalin injection (Bhutta et al., 2001), but was increased f ollowing repeat needle stick injury (Anand et al., 1999). Tissue analysis and mechanisms An obvious advantage of laboratory studies is the ability to ana- lyse tissue at various sites to evaluate both the impact of the initial injury and an y long-term consequences in structure and/or func- tion. Examples include: CHAPTER 3 long-term effects of early pain and injury: animal models 23 Maternal separation, handling, and neonatal stress As developmental studies involve experim ents on young animals that are dependent on maternal care, it is necessary to control for n on-injury related eff ects due to handling and separation or inter- actions with the mother (Sternberg and Ridgway, 2003). injection of carrageenan in diff erent volumes and concentrations (0.5 \200\2232 mcl/g of 0 .25 \200\2232%) produces a robust but shorter duration acute infl ammatory hyperalgesia (Re n et al., 2004; Torsney and Fitzgerald, 2002; S. M. Walker et al., 2003), and doses as low as 1 mcgl/g of 0.25% lambda carrageenan produce persistent eff ects (Anseloni et al., 2005; Ren et al., 2004; G. Wang et al., 2004). Impact on sensory thresholds Large volumes of CFA in neonatal rodents (25 mcl in P1 rat or 20 mcl in P1 mouse) have been associated with either no change (Lim et al., 2009; Ruda et a l., 2000; S. M. Walker et al., 2003) or a reduction in sensory thresholds (i.e. increased s ensitivity) (Blom et al., 2006; Roizenblatt et al., 2010) in adulthood. By contrast, carrag eenan (1 mcl/g 0.25%) has been consistently associated with higher thresholds (i.e. decreas ed sensitivity) from P30 until adulthood (Anseloni et al., 2005; Lidow et al., 2001; Ren et al., 2004; G. Wang et al., 2004). In addition to a generalized hypoalgesia in all paws, ne onatal carrageenan also decreased the visceromo- tor response to colorectal distension in a dulthood, suggesting a modulatory impact on both somatic and visceral processing (G. Wang e t al., 2004). Repeated and oft en prolonged maternal separation, for up to 3 hours a day across the fi rs t 2 postnatal weeks, increases visceral sensitivity in adulthood (Chung et al., 2007; Schwe tz et al., 2005; Tjong et al., 2010). Alterations in subsequent stress-induced analge- sia (Sternberg and Ridgway, 2003) and opioid tolerance (Kalinichev et al., 2002) suggest change s in endogenous pain inhibitory systems. Th is degree of handling and duration of separatio n is predominantly utilized to assess changes in stress-related behavior mediated via the h ypothalamic \200\223pituitary \200\223adrenal axis (Chung et al., 2007; Faturi et al., 2010 ), and is greater than required for most neonatal injury models. However, control groups wi th the same degree of handling and separation in the absence of the noxious stimulus are re quired to confi rm that eff ects seen in later life are specifi c for the injury itself. Th e impact of maternal \200\223pup interactions is complex, as injury in the pups may also a lter maternal behavior and outcomes. An initial increase in maternal contact but later negl ect was reported aft er hindpaw infl ammation in rat pups (Anseloni et al., 2005), but no c hange in maternal behavior was seen following laparotomy in newborn mice (Sternberg and Rid gway, 2003; Sternberg et al., 2005). Maternal care may modulate adverse eff ects: increased grooming was noted aft er P6 and was postulated to decrease the impact of repeated heel pr ick from P2 to P14 in rats (C. D. Walker et al., 2003). In a subsequent study, varying leve ls of grooming by diff erent mothers infl uenced thermal withdrawal latency in adult- hood, but had no impact on long-term eff ects following repeated formalin injection (twice daily from P3 to P14; C. D. Walker et al., 2008). Th e need to care for injured pups may also al ter maternal outcomes, with one study reporting long-term alterations in pain threshold, an xiety, and sleep fragmentation in the dams of pups with CFA-induced hindpaw infl ammation ( Roizenblatt et al., 2010). Response to subsequent noxious stimulus Neonatal hindpaw infl ammation increases the respon se to noxious stimuli in adulthood. CFA 25 mcl at P1 enhanced the response to diff erent st imuli in the previously injured paw including: increased behavioral hyperalgesia following repeat CFA (Lim et al., 2009; Ruda et al., 2000) or capsaicin (Hohmann et al., 2005); earli er onset of response to formalin; and increased dorsal horn neuronal response to peripheral brush or pinch (Ruda et al. 2000). Prior CFA infl ammation did not alter the response to n erve injury in adult- hood (Lim et al., 2009). Neonatal carrageenan infl ammation also incr eases the degree of behavioral hyperalgesia to subsequent infl ammation (Ren et al., 2004) or incision (Chu et al., 2007) of the previously injured paw. Critical period Th e fi rst postnatal week is the critical period for persistent eff ects f ollowing infl ammation. Persistent changes follow CFA 25 mcl at P1, but not 50 mcl CFA at P 14 in rats (Hohmann et al., 2005; Ruda et al., 2000) and CFA 20 mcl at P1 but not 40 mcl at P14 in mice (Blom et al., 2006). However, it is diffi cult to determine if these vol- umes produced the same degree of infl ammation at the two ages. Carrageenan in doses adjusted f or body weight (1 mcl/g) at P0, P1, P3, and P5 but not at P8 and older was associated with long-term alterations in threshold (Ren et al., 2004), and the pattern of gene expression i n the dorsal horn also diff ered if injury occurred at P3 rather than at P12 (Ren et al., 2 005). Similarly, altered behaviors indicative of a low anxiety trait in adulthood were seen follow- ing carrageenan infl ammation at P3 but not P12 (Anseloni et al., 2005). Evaluating the long-term impact of early pain: speci \201 c injury models A large number o f diff erent injury models have evaluated the impact of early pain, and responses vary both with the type and severity of initial injury (Fitzgerald and Walker, 2009) (Figure 3.1 ).