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 ). Peripheral in \202 ammation Initial injury Diff erent agents can induce a peripheral infl ammatory response, are commonly injected in the hindpaw, and changes in hindlimb refl ex se nsitivity assess the degree of subsequent sensitivity. CFA comprises inactivated mycobacter ia emulsifi ed in mineral oil, and carrageenan contains polysaccharides extracted from seaw eeds. Mechanisms Injection of CFA 25 mcl in newborn rat pups produces long-term increases in paw diameter (not seen aft er carrageenan or smaller volumes or CFA) and histological signs of chronic infl ammation (Lim et al., 2009; S. M. Walker et al., 2003). Th is may contribute t o the persistent reductions in sensory threshold that have been reported following this sti mulus (Blom et al., 2006; Roizenblatt et al., 2010). Long-term eff ects following neonatal infl ammation vary with the type and severity of stim ulus. An early model documenting persist- ent changes in sensory processing utilized 25 mcl CFA on P1 (Ruda et al., 2000), however this is a relatively large volume in the neonatal p aw, producing chronic infl ammation rather than a specifi c neo- natal insult (Lim et al., 2009; S. M. Walker et al., 2003). Hindpaw SECTION 1 introduction 24 A Repeat needle injection \200 Hyperalgesia Repeat formalin injection \200 Hypoalgesia Spinal nerve lig ation \200 Minimal allodynia Chronic constriction injury \200 No allodynia Partial sciatic lig ation \200 No allodynia H+ Spared nerve injury \200 No allodynia \200 No microg lial respo nse in spinal cord IM low pH injection \200 Visceral hyper alg esia \200 Hyper alg esia to muscle pinch Full-thickness skin wound \200 Hyperinner vation \200 Localized hyperalgesia B Hindpaw inflammation \200 Generalized hypoalg esia \200 Increased response to repeat in jury (inflammation, incision) Bowel inflammation \200 Increased sensitivity to distension in adulthood Bladder inflammation \200 Increased sensitivity to inflammation in adulthood \200 Incre ased spontaneous micturition frequency LPS Immu ne challeng e (IP lipopolysacchar ide) \200 Baseline hyper alg esia Abdominal incision \200 Thermal hypoalg esia \200 Decreased response to IP acetic acid Plantar incision \200 Enhanced respo nse to repeat incision Figure 3.1 Diagramatic representation of di \200 erent early injuries and associated impac t on long-term sensory outcomes. See text for further details. IM, intramuscular; IP, intra peritoneal. Reprinted by permission from Macmillan Publishers Ltd. From Fitzgerald, M. and Walker, S. M., Infant pain management: a developmental neurobiological approach, Nature Cli nical Practice Neurology , Volume 5, Issue 1, pp. 35 \200\22350, Copyright 2009, http:// www.nature.com/nrneurol/index.html As the baseline hypo algesia following carrageenan is generalized to all paws and emerges l ater in development (aft er P30), brain- stem-mediated alterations in descending modulation have been postulated (G. Wang et al., 2004). Enhanced inhibition from the rostroventral me dulla (RVM; Zhang et al., 2010) and alterations in opioid-mediated response in the periaque ductal gray (PAG; Laprairie and Murphy, 2009) have been noted in adults following neonatal hindpaw carrageenan. Increased expression of several subtypes of serotonin (5HT) receptor i n the PAG has also followed early infl ammation (Anseloni et al., 2005). Enhanced re-injury responses are apparent from soon aft er the initial injury and are segme ntally restricted to the previously infl amed paw (Chu et al., 2007; Ren et al., 2004). Fol lowing CFA 25 mcl at P1 (Ruda et al., 2000) (but not CFA 5 mcl or 25 mcl carrageenan; S. M. Walker et al., 2003), the terminal fi eld of sciatic nerve aff erents was expanded in the ipsilateral spinal cord, with increased calcitonin gene-related peptide-positive terminals. Th is likely contributes to an enhanced central response to a noxious peripheral stimulus, as supported by increased c-fos expression in response to repeat CFA (Tachibana et al., 20 01) or capsaicin in adulthood (Hohmann et al., 2005). Long-term alterations in the spinal c ord expression of genes related to nocicep- tive transmitter systems (Ren et al., 2005), an d in the function and/ or structure of N-methyl-D-aspartate receptors in the spinal cord Diff erent mechanisms are likely to underlie the enhanced hyper- algesic response to noxiou s stimuli in adulthood, which diff ers in both time course and distribution from changes in baseline threshold. CHAPTER 3 long-term effects of early pain and injury: animal models 25 Impact on sensory responses A 25-gauge needle was inserted through each paw, once, twice, o r four times daily from P0 to P7. Th ermal sensitivity was increased in the four-times dail y injection group at P16 and P22. Th is may refl ect residual infl ammation, as there was n o signifi cant diff erence from controls by P65 (Anand et al., 1999). (Chu et al., 2007) and in the thalamus and other brain regions (Blom et al., 2006) may also contribute to enhanced responses to subsequent injury. Modulation by analgesia Th e long-term impact of neonatal carrageenan infl ammation was red uced by morphine at the time of the initial insult (Laprairie et al., 2008; Rahman et al., 1997). Repeated unilateral injections in the gastrocnemius muscle on alternate days from P8 to P20 were performed with a needle alone or combined with injection of 100 mcl of an acidic pH 4 or physiologi- cal pH 7.4 saline solution. No intervention altered refl ex sensitivity to a hindpaw mechanical stimulus in adulthood (Miranda et al., 2006). Formalin injection Initial injury Injection of formalin (0.5 \200\22315%) into the hindpaw is used as a model of acute nociception and produces a biphasic behavioral response with li cking, twitching, fl icking, and elevation of the paw in adult rats (Le Bars et al., 2001). Although responses diff er in neonatal rat pups, formalin does produce specifi c nocicepti ve behaviors that are reduced by morphine (Abbott and Guy, 1995; Barr et al., 2003; McLaugh lin et al., 1990). Response to subsequent noxious stimulus Although there were no detectable changes in muscle histology, the response to deep muscle pinch was enhanced following repeated muscle inject ion of the pH 4, but not pH 7, solution. Additionally, the pH 4 group had an increased visc eromotor response (EMG response in abdominal muscles) and spinal dorsal horn neuron respons e (in areas of viscerosomatic convergence) to colonic dis- tension in adulthood, which may refl ect more generalized altera- tions in descending modulation (Miranda et al., 2006). Impact on sensory thresholds Repeated injection of 5 mcl of 10% formalin (all four paws at P1 and forepaws or hindpaws on alternate days from P2 to P7; Bhutta et al., 2001) or daily injections into each hindpaw from P1 to P4 (5 mcl of 4%; Anand et al., 2007; Rovnaghi et al ., 2008) decreased thermal sensitivity in adulthood. As responses were compared to undistur bed controls, the impact of repeated injection versus the formalin injectate itself cannot be determined. By contrast, for- malin at a single time point (10 mcl of 4% into right fore paw and hindpaw on P1) had no impact on thermal latency in adulthood (Negrigo et al., 2011) . Twice-daily injections of a lower concentra- tion of formalin (10 mcl 0.2 \200\2230.4%) i n alternating hindpaws from P3 to P14 also had no impact on thermal sensitivity in adulthoo d (C. D. Walker et al., 2008). Full-thickness skin wound Initial injury Neonatal skin is thin and sensitive to injury (Fox , 2011). In the lab- oratory, long-term eff ects have been evaluated following removal of a small full-thickness skin fl ap on the dorsal hindpaw (Reynolds and Fitzgerald, 1995). Impact on sensory responses Neonatal skin wounding produces localized hyperalgesia that per - sists well beyond the period of wound healing (Alvares et al., 2000; Reynolds and Fitzger ald, 1995; Young et al., 2008). Response to subsequent noxious stimulus Repeated formalin from P3 to P14 increased the area under the behavioral response curve following subsequent hindpaw forma- lin in adulthood ( C. D. Walker et al., 2008). Critical period Persistent hyperalgesia follows full-thickness skin wounding in the fi rst postnatal week, but not at older ages, and the degree of hyper- innervation is much greater than seen following the same injury in adulthood (Reynolds and Fitzgerald, 1995). Critical period and mechanisms Th ere has been limited comparison of age-dependent suscepti - bility for persistent eff ects following formalin, as many protocols include repeated inj ections across the fi rst 1 to 3 postnatal weeks. Histological evaluation of the eff ect of repeated formalin injection on the peripheral tissue would be benefi cial to exclude chron ic injury. Th e pattern of gene expression in the spinal cord diff ered following hindpaw f ormalin injection at P3 (5 mcl or 20 mcl of 2%) versus P21 (200 mcl of 2%; Barr et al., 200 5). Mechanism Full-thickness skin wounds result in both peripheral changes in innervation densi ty and central changes in the spinal cord. Marked peripheral hyperinnervation due to collat eral sprouting from adja- cent axons, and from distant intact neurons (Alvares and Fitzgera ld, 1999), occurs within the wounded area, mediated by local release of neurotrophic factor s including NGF and NT-3 (Beggs et al., 2012a; Constantinou et al., 1994; Reynolds et al., 1997). In addi- tion, there are changes in short-range neural inhibitory cues (Moss et al., 2005). Ephrins act as contact-mediated guidance molecules during development and repair, a nd ephrin-A4 inhibits neurite out- growth. However, following neonatal skin wounding this i nhibition is reduced as ephrin-A4 expression is downregulated, allowing an increase in inne rvation density (Moss et al., 2005). NK-1 expressing neurons in the spinal cord are involve d as ablating these neurons with intrathecal substance P-saporin at the time of neonatal in jury or in adulthood reduced injury-related eff ects (Young et al., 2008). In addition, the receptive fi eld size of dorsal horn neurons is signifi - cantly greater 3 and 6 weeks fol lowing neonatal skin wounding when compared with control animals (Torsney and Fitzgerald, 2 003). Response to analgesia Long-term behavioral changes in adults were reduced by mor- phine adm inistration prior to neonatal formalin injection (Bhutta et al., 2001). Ketamine reduced th e thermal hypoalgesia in adult females, but not in males (Anand et al., 2007). Repeat needle injury Initial injury Repeated needle injection in the paws of rat pups has b een per- formed to model repeated heel lance for clinical blood sampling in neonates. Howev er, the relative size of the needle and depth of penetration may be relatively greater in t he rodent paw. SECTION 1 introduction 26 Modulation by analgesia A single sciatic block with bupivacaine prior to skin wounding prod uced an acute sensory and motor block but did not prevent hyperinnervation or persistent hy peralgesia (De Lima et al., 1999). Intra-articular injection of lidocaine in the medial and lateral ankle reduced acute vocalizations following stimulation of the injured hindpaw, al though it is not clear how frequently injections were performed, and there was no impact on persistent eff ects (Young et al., 2007). Visceral injury Initial injury Visceral injury is modeled in rodents by exposing the bladde r or to bowel to chemical irritants, such as mustard oil or zymosan, or repeated mechanical stimuli (e.g. rectal distension). Impact on sensory responses and future injury Early visceral injury has been associated wit h visceral hyperalge- sia in adulthood. Daily mechanical distension or chemical (mus- tard oil 0.2 ml 5%) colonic irritation from P8 to P21 increased behavioral (Al-Chaer et al., 200 0) and abdominal muscle EMG responses to subsequent colonic distension, and also altered fo od consumption and fecal output (J. Wang et al., 2008). Shorter exposures to intracolonic m ustard oil (2% 10 mcl from P8 to P10; Christianson et al., 2010) or infusion of acetic acid at P10 (Winston et al., 2007) also increased sensitivity to mechanical colonic disten- sio n in adulthood. Surgical incision Initial injury Surgical injury produces infl ammation, skin wounding, and periph- eral nerve injury (Kehlet et al., 2006). Th e plantar hindpaw incision model compr ises skin incision plus elevation and incision of the underlying plantaris muscle, and prod uces acute hyperalgesia and increased hindlimb refl ex sensitivity at all postnatal ages in the rat (Ririe et al., 2003; S. M. Walker et al., 2009). A midline abdominal incision in m ice on the day of birth has also evaluated the impact of neonatal laparotomy (Sternberg et al., 2005). Bladder infl ammation (daily intravesical injections of zymosan P14 \200\223P16) increased spontaneous micturition frequency and the response to subsequent infl ammation in adulthood (Randich et al., 2006). Impact on sensory responses and future injury Neonatal laparotomy in mice was associated wi th a generalized decrease in baseline sensitivity to thermal and visceral stimuli in adulth ood (Sternberg et al., 2005). Similarly, and as seen following carrageenan infl ammation, n eonatal hindpaw incision decreases baseline sensitivity to mechanical and thermal stimuli i n adult- hood, but a subsequent injury unmasks segmental hyperalgesia. Both the degree and duration of hyperalgesia are increased when the same paw is re-injured, either 2 weeks late r or in adulthood (P60) (Beggs et al., 2012b; S. M. Walker et al., 2009). A series of three hindpaw incisions performed at P3, P10, and P17 also increased the degree of incision-rela ted hyperalgesia at P90 (Low and Fitzgerald, 2012). Spinal neurons receive convergent input from both somatic and visceral aff erents, and neon atal visceral injury increased somatic refl ex sensitivity to hindpaw thermal (J. Wang et a l., 2008) or mechanical stimuli (Christianson et al., 2010), in some but not all models (Ra ndich et al., 2006). Neonatal somatic insults also have an impact on visceral sensitivity i n adulthood, as noted in previous sections (Miranda et al., 2006; G. Wang et al., 2004). Critical period Visceral insults in the fi rst 2 to 3 postnatal weeks, but not at older age s, have been associated with persistent changes in sensitivity, including: repeat colonic s timulus from P1 to P20 but not P21 to P42 (Al-Chaer et al., 2000); daily bladder injections from P14 to P16 but not P28 to P30 (Randich et al., 2006); intracolonic acetic acid at P10 but not 8 weeks of age (Winston et al., 2007). Critical period Hindpaw incision in the fi rst postnatal week (P3 or P6) but not at older ( P10, P21, P40) ages enhanced the response to future incision (S. M. Walker et al., 2009). Mechanism Neonatal colonic irritation increased electrophysiological responses in adult spi nal cord viscerosensitive neurons, in the absence of any demonstrable changes in peripheral bowel histology (Al-Chaer et al., 2000; Christianson et al., 2010; Lin and Al-Chaer, 2003) . Changes in receptor expression in the DRG may refl ect the nature of the initial insult. Increased TRPV1 expression in cell bodies of colon aff erents in the DRG followed acetic ac id irritation at P10 (Winston et al., 2007); whereas P8 to P10 mustard oil (which stimu- la tes TRPA-1 receptors), increased expression of TRPA-1 but not TRPV1 (Christianson et al., 2 010). Mechanism Prior neonatal incision primes the spinal neuroimmune response to repeat incision in adulthood. Th e enhanced incision-related hyper- algesia in adults with prior neonatal incision is associated with an earlier onset, and increased duration and degree of microgli al reac- tivity in the spinal cord (Beggs et al., 2012b). Modulation by analgesia Administration of systemic morphine at the time of neonatal laparot omy prevented long-term changes in sensory responses (Sternberg et al., 2005). Th e eff ect of neonatal incision on later inci- sion-related hyperalgesia was prevented by repeated pe rioperative sciatic blocks (four 2-hourly blocks) but not by a single preopera- tive block, suggesting that more prolonged primary aff erent block- ade is required to modify persiste nt eff ects. In adults with prior neonatal incision, intrathecal minocycline selectively pr events the enhanced hyperalgesic response, confi rming a functional role for microglial rea ctivity (Beggs et al., 2012b; see Vega-Avelaira and Beggs, Chapter 7, this volume, for furt her details of neuroimmune interactions in nociceptive pathways). Immune challenge Initial injury Early life immune challenges are modeled by various stimuli , such as injection of the bacterial endotoxin lipopolysaccharide (LPS) or endothelin-1, an d can have a signifi cant impact on the immune response in later life (McKelvy and Sweitzer , 2009; Spencer et al., 2011). Impact on sensory responses and mechanisms Intraperitoneal LPS at P14 had persistent eff ec ts on sensory function, as hindpaw sensitivity to mechanical and thermal stimuli and spinal CHAPTER 3 long-term effects of early pain and injury: animal models 27 expression of cyclo-oxygenase 2 was increased in adulthood (Boisse et al., 2005). Similarly , intracerebral injection of LPS at P5 produced persistent hyperalgesia and increased micro glial reactivity in the brain, which was attenuated by administration of an interleukin-1 r eceptor antagonist (K. C. Wang et al., 2011). As severe infl amma- tion may be associated w ith a signifi cant immune response, and increased microglial reactivity in the spinal cord has been associated with persistent alterations in pain sensitivity, interactions between t he immune system and nociceptive processing can have a signifi cant impact on the long-term consequences of early injury. Anseloni, V. C., He, F., Novikova, S. I., Turnbach Robbins, M., Lidow, I. A., Ennis, M., et al . (2005). Alterations in stress-associated behaviors and neurochemical markers in adult rats aft er neonatal short-lasting local infl ammatory insult. Neuroscience, 131 , 635 \200\223645 . Back, S. K., Kim, M. A., Kim, H. J., Lee, J., Sung, B., Yoon, Y., et al . (2008). Developme ntal characteristics of neuropathic pain induced by peripheral nerve injury of rats during neonatal period. Neurosci Res, 61 , 412 \200\223419 . Barr, G. A., Gao, P., Wang, S., Cheng, J., Qin, J., Sibille, E. L., et al . (2005). Microar ray analysis of gene expression following the formalin test in the infant rat. Pain, 117 , 6 \200\22318 . Barr, G. A., Limon, E., Luthmann, R. A., Barr, G. A., Cheng, J., and Wang, S. (2003). Analg esia induced by local plantar injections of opiates in the formalin test in infant rats. De v Psychobiol, 42 , 111 \200\223122 . Peripheral nerve injury Several diff erent models have been used to investigate the patho- physiology of traumatic nerve injury and ontogeny of neuropathic pain (Sorkin and Yaksh, 20 09). In rodents, injury to the sciatic nerve or its branches or related central spinal nerv es allows evalu- ation of changes in hindlimb refl ex responses to hindpaw sensory stimuli, and comparison both with baseline and the contralateral side. Spared nerve injury, chronic constriction injury, partial sciatic ligation, and spinal nerve ligation performed in the fi rst 2 to 3 weeks of postnatal life do not produce the same degree of acute allodynia as seen in adults (Howard et al., 2005; Lee and Chung, 1996; Ririe and Eisenach, 2006). Less c ommonly, nerves innervating the tail are injured and tail fl ick responses are used to eval uate sensitivity (Back et al., 2008). Age-related diff erences in the glial response to ner ve injury contribute to the lack of neuropathic allodynia in early life. Th e eff ects and mechanisms of nerve injury are discussed by Vega-Avelaira and Beggs (Chapter 7, this volume ). Beggs, S., Alvares, D., Moss, A., Currie, G., Middleton, J., Salter, M.W., and Fitzgerald, M (2012a). A role for NT-3 in the hyperinnervation of neonatally wounded skin. Pain, 153 , 2133-2139 . Beggs, S., Currie, G., Salter, M. W., Fitzgerald, M., and Walker, S. M. (2012b). Priming of adult pain responses by neonatal pain experience: maintenance by central neuroimmune activ ity. Brain, 135 , 404 \200\223417 . Bhutta, A. T., Rovnaghi, C., Simpson, P. M., Gossett, J . M., Scalzo, F. M., and Anand, K. J. (2001). Interactions of infl ammatory pain and morphi ne in infant rats: long-term behavioral eff ects. Physiol Behav, 73 , 51 \200\22358 . Blom, J. M., Benatti, C., Alboni, S., Capone, G., Ferraguti, C., Brunello, N., et al . (2006). E arly postnatal chronic infl ammation produces long-term changes in pain behavior and N-meth yl-D-aspartate receptor subtype gene expression in the central nervous system of adult mice . J Neurosci Res, 84 , 1789 \200\2231798 . Boisse, L., Spencer, S. J., Mouihate, A., Vergnolle, N., and Pittman, Q. J. (2005). Neonata l immune challenge alters nociception in the adult rat. Pain, 119 , 133 \200\223141 . Brambrink, A. M., Evers, A. S., Avidan, M. S., Farber, N. B., Smith, D. J., Zhang, X., et a l . (2010). Isofl urane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesth esiology, 112 , 834 \200\223841 . Conclusion Both clinical and preclinical studies demonstrate the complex- ity and diversity of persistent changes in pain responses following early pain and injury. It is too simplis tic to expect that pain experi- ence will reliably either increase or decrease sensitivity, as multiple contributory factors may interact to infl uence nociceptive process- ing and/o r the behavioral response to pain. Evaluation in animal models is essential to investigate age-dependent changes in both the acute and persistent eff ects of diff erent forms of inju ry, understand underlying mechanisms, and ultimately how eff ects may be modi- fi ed or pre vented by clinically applicable analgesic interventions. Cataldo, G., Bernal, S. Y., Rozengurtel, S., Medina, K., and Bodnar, R. J. (2010). Neonatal and adult gonadal hormone manipulations enhance morphine analgesia elicited from the ventr olateral periaqueductal gray in female rats. Int J Neurosci, 120 , 265 \200\223272 . Christianson, J. A., Bielefeldt, K., Malin, S. A., and Davis, B. M. (2010). Neonatal colon insult alters growth factor expression and TRPA1 responses in adult mice. Pain, 151 , 540 \200\223549 . 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