e T lymphocytes which are present in the dorsal root ganglion (DRG), and increased activity and recruitment fol- lowing nerve injury can contribute to pain sensitivity (Hu et al., 20 07). In the spinal cord, microglia are no longer thought of as having only immune surveilla nce and supportive roles. Following noxious aff erent input, microglial reactivity is incre ased, and the released proinfl ammatory cytokines, chemokines, and extracellu- lar protease s, interact with neurons and contribute to central sen- sitization (Beggs and Salter, 2010; Clark et al., 2011). Astrocytes, another subtype of glial cells, are also activated in the spinal cord following infl ammation, plantar incision, and nerve injury, but the degree an d time course diff er from microglia, and varies with the type of injury (Chiang et al., 20 12). Modulators of glial activ- ity reduce pain sensitivity associated with injury, but hav e little eff ect on normal or baseline pain processing, suggesting that the main role of gl ial activation is in pathological pain states (Chiang et al., 2012). PNI in experimental animals under 3 weeks of age does not result in the development of pain hypersensitivity, although evidence sug- gests that the onset is delayed (Howard et al., 2 005; Vega-Avelaira et al., 2012). Th is developmental anomaly is due, at least in part, to diff erences between the early postnatal and adult immune systems (Moss et al., 2007; Vega- Avelaira et al., 2007, 2009). Microglia Microglia are the resident macrophages of the central nervous system (CNS). Th ei r complex repertoire of functions in the adult includes maintaining homeostasis, detecting and responding to infection, and promoting or suppressing neuroinfl ammation. Th ese action s also link microglia inextricably to neurodegenera- tive diseases such as Alzheimer \200 \231s disease, Parkinsonism, and neuro- pathic pain (Beggs et al., 2012b; Kettenmann et al. , 2011). Microglia respond to potentially damaging injury or disease to the CNS by adopting any of several reactive phenotypes. Morphological changes can be identifi ed by changes in expression of the microglial marker, ionized calcium binding adaptor molecule1 (Iba-1; Beg gs and Salter, 2007; Suter et al., 2009; Vega-Avelaira et al., 2007). Where once the concep t of a monolithic microglial \200\230activation \200\231 was widely accepted, evidence now suggests there are diff erent subpopulations SECTION 2 biological basis of pediatric pain 66 DRG SC Periphery Resting microglia CCL2 \206\222 M \206, \206\221 CCL2, \206\221 M \206, Mastocytes \206\221 Proin \202ammatory cytokine \206\221 M \206, Mastocytes \206\221 Action Potentials 1ry a \200erent ATP CCL2 P 2 X 4 C C R 2 NF- B IL-Rs p38 mRNA \206\221 N M D A R N K 1 R A M P A R BDNF PGs TNF IL-6 IL-1 Activated microglia 2nd order neuron Figure 7.1 Neuroimmune interactions in neuropathic pain. Peripheral nerve injury provokes a ctivation of innate immune cells such as macrophages and mastocytes proximal to the damaged area (Hu and McLachlan, 2002, 2003; Watkins and Maier, 2002). As a consequence, both immun e cells and Schwann cells secrete pro- in \202 ammatory cytokines to prevent further injur y and repair the damage. Th is promotes physiological, cellular, and molecular changes in t he DRG neurons and macrophages which result in neuronal aberrant electrical discharges, mac rophage activation and pro-in \202 ammatory cytokine release (Gabay et al., 2011; Hu and M cLachlan, 2002; Ozaktay et al., 2006; Suter et al., 2009). Cytokines, such as CCL2 are ante rogradely transported from the ganglia to the primary a \200 erents terminals in the spina l cord (Th acker et al., 2009). Acting through CCR2 and interleukin receptors they activate intracellular pathways (e.g. P38 mak kinase pathway), promote translocation of transcripti on factors like NF-kB to microglia nuclei, and become activated (Capiralla et al., 2012; Ch ang and Waxman, 2010; Ren and Dubner, 2007; Sch fers et al., 2003; Svensson et al., 2003 ). As part of the activation process, microglia produce and release cytokines with an autoc rine and paracrine activity which create a feedback positive loop that will sensitize secon d-order neurons and initiate the neuropathy (X. Gao et al., 2005; Y.-J. Gao and Ji, 2009, 2 010). with diff erent reactive response profi les and capabilities (Scheff el et al., 2012). development are complex with a dual function as: (1) immune eff ec- tors in the removal of excess and exuberant axonal projections, and (2) a more classical glial role as cytotropic and cytotrophic factors (Schlegelmilch et al., 2011). Given the extensive remodeling and re fi ning of connectivity throughout the CNS in the early postnatal period, it is clearly nec essary for microglia to diff erentiate between normal physiology and pathology. Microglia are sensors for damage or disease in the CNS, capa- ble of detecting pathogen-ass ociated molecular patters (PAMPs) and damage-associated molecular patterns (DAMPs). One of the many classes of receptors expressed by microglia are the Toll-like receptors (TLRs). TL Rs are part of the complex molecular sensor framework for detecting both PAMPs and DAMPs (S tewart et al., 2010; Zhang and Mosser, 2008) and members of this family of receptors have b een implicated in the pathogenesis of neuropathic and infl ammatory pain in adult animals ( Sorge et al., 2011; Tanga et al., 2005). Th e induction of cytokines and chemokines resulting from TLR4 activation varies with postn atal age and the pattern is the same across the TLR family members, with a general hypo res ponsiv- ity that peaks at postnatal day (P) 21. Th e transient changes and hyporesponsivity of some TLRs may be a refl ection of the substan- tial reorganization and refi nement of c onnectivity within the dorsal horn (and CNS generally) that occurs postnatally. Normal deve l- opmental synaptic elimination and pruning of exuberant axonal projections would provide a temporary increase in DAMPs and provoke an unwanted immune response. Microglial function during development Microglia are of haematopoietic origin, and fi rst a ppear as primi- tive macrophages in the yolk sac in early embryogenesis from where they mig rate into the CNS (Ginhoux et al., 2010). Th is process occurs throughout embryonic and int o early postna- tal life (Pont-Lezica et al., 2011). Th e actions of microglia during Th is time point marks the end of a critical period of matura- tion of both the nervous and immune systems, and also the point CHAPTER 7 neuroimmune interactions and pain during postnatal development 67 at which central neuroimmune interactions stimulated by PNI are capable of driving neuropat hic behavioral pain behaviors (Howard et al., 2005; Moss et al., 2007). Intriguingly, this postnatal maturation is also marked by a switch in gene expression such that gene transcrip ts associated with the classical, pro-infl ammatory M1 phenotype are downregulated while th ose of the alternative anti-infl ammatory M2 phenotype start to predominate (Gordon and Tay lor, 2005; Mosser and Edwards, 2008; Scheff el et al., 2012). an amoeboid morphology and maximal proliferation, and persists for at least 3 months (Hu an d McLachlan, 2002). Retrograde label- ling studies have shown that these reactive macrophag es cluster around damaged neurons and in particular the large cell bodies of myelinated aff erents (Vega-Avelaira et al., 2009). Th e injury-induced reactive phenotype of DRG macrophages is determined in part by their mo lecular phenotype. Increased expres- sion of membrane receptors such as purinergic receptor s (e.g. P2X 4 ) and chemokine receptors (e.g. IL6R, TNF R, CCR2) activate intra- cellula r second-messenger pathways (e.g. MAP kinases pathway; Gao and Ji, 2010; Ji et al., 2003, 2 009; Scholz and Woolf, 2007), lead- ing to increased expression of molecules that drive a p hagocytic phenotype. Th is is accompanied by the release of chemokines (Hu and McLachlan, 2 003) which maintain this adaptive phenotype through both autocrine and paracrine positive f eedback loops and by macrophage recruitment from the circulation. Chemokines also act direc tly to infl uence neuronal excitability (Scholz and Woolf, 2007), thereby modulating aff er ent signal transduction from the dorsal root ganglia to the spinal cord. T lymphocytes T cells, also known as T lymphocytes, play an important role in cell- mediate d immunity, but there is also increasing evidence for a role of T cells in the responses to PNI that produce pain. Helper T cells can be divided into two broad subtypes; Th 1 and Th 2, classifi ed as such by the array of cytokines they produce. Th 1 is generally pro- infl ammatory and released cytokines include interferon gamma (IFN ), which is heavily implic ated in neuropathic pain pathogen- esis (Costigan et al., 2009; Masuda et al., 2012; Tsuda et al., 2009). Th 2 is generally anti-infl ammatory, and ideally a balance of Th 1 and Th 2 exists. In early life, the Th 1 pro-infl ammatory response is weak (Fadel and Sarzotti, 20 00), and the balance is shift ed to a Th 2 predominance, with Th 1 gradually strengthening with exposure to allergens. Signaling molecules MCP-1/CCL2 Macrophage chemo-attractant protein 1 (MCP-1, now CCL2) and its receptor CCR2 have been associated with several models of nerve injury (Th acker et al. , 2009; Vega-Avelaira et al., 2009). MCP-1 is expressed in small DRG neurons and is transpo rted anterogradely to the spinal cord aft er PNI where it has chemotactic eff ects on both glia and neurons (Th acker et al., 2009) (Figure 7.1). Genetically modifi ed CCR2 null or \200\230knock-out \200\231 mice do not exhibit enhanced pain behavior aft er PNI (Abbadie e t al., 2003; Zhang et al., 2007). Taken together, these studies directly implicate macro- p hage-neuronal interactions in pain behavior. Th e complex developmental regulation of the immune system is occurring at the same time as the nervous system is undergo- ing extensive postnatal refi nement (Beggs et al., 2002; Fi tzgerald, 2005; Fitzgerald et al., 1994), it is not surprising that interactions of the imm une system with the CNS following PNI diff er signifi - cantly in the early postnatal perio d from those seen in the adult. Taken together, these observations indicate that the immune sys- tem in the immediate postnatal period does not react to PNI in the same way as in the mature adult animal. Th e pattern of T-cell acti- vation and infi ltration following PNI d iff ers signifi cantly in young animals. Th e predominance of Th 2 T cells in the neonate u nderlies this, imbuing the immature immune system with a tolerance for the new antigens exp osed by normal developmental changes within the CNS (Costigan et al., 2009). Tumor necrosis factor (TNF ) TNF is a pro-infl ammatory cytokine and one of many f ac- tors released following PNI (Jeon et al., 2011; Kim et al., 2011). Macrophages are the main source of TNF but de novo expres- sion is also evident in small-diameter nociceptiv e cells aft er nerve injury. TNF activates the MAP kinase p38 intracellular pathway whic h instigates a feed-forward mechanism to increase further production of TNF . Pharmacolo gical blockade of TNF is suffi - cient to ameliorate pain behavior in animals with parti al axotomy (Sch fers et al., 2003). Neuroimmune interactions in dorsal root ganglia Macrophages are phagocytic immune cells, ca pable of engulfi ng and digesting cellular debris and pathogens, and act to maintain the ce llular environment and protect against potential infection in the DRG (Watkins and Maier, 2 002). In keeping with resident microglia in the CNS, macrophages in the DRG adopt a ramifi ed surveillance state and are homogenously distributed between neuronal cell bod- ies and p rojecting fi bres (Beggs et al., 2012b; Hu and McLachlan, 2002, 2003; Vega-Avelaira et al., 2009). PNI provokes both a reac- tive response of the resident macrophages as well as infi ltration of new macrophages from the circulation (Hu and McLachlan, 2002, 2003). Macrophag es in the DRG are characterized by expression of the \200\230macrophage scavenger receptor \200\231 CD163, and can also be iden- tifi ed by expression of CD68, which is increased 3 days following experimental PNI in adult animals (Hu et al., 2007). CD168-positive cell num ber remains the same, suggesting a reactive phenotypic switch in the normal resident macrop hage population. Macrophage reactivity peaks at 7 days post injury, as shown by the adoptio n of Interleukin-6 (IL-6) IL-6 is a pleiotropic cytokine (Simpson et al., 1997) with roles in ne uronal survival, growth and diff erentiation (Grothe et al., 2000). It is synthesized and r eleased by macrophages and Schwann cells. Th e IL-6 receptor (IL-6R or CD126) is expressed in the DRG (Br zda et al., 2009; Watkins and Maier, 2002; Zhang et al., 2007) and there is evidence for IL-6 involvement in both the hyperalgesia and allodynia associated with neu ropathic pain (DeLeo et al., 1996). IL-6 production is increased in neurons and satellite g lial cells in DRGs within 24 h of PNI and persists for at least 2 weeks (Br zda et al., 2009). Furthermore, IL-6 has roles in multiple independent pathways mediating processes inc lud- ing nerve regeneration (Qiu et al., 2005) and TNF -induced SECTION 2 biological basis of pediatric pain 68 apoptosis of DRG neurons (Murata et al., 2011). As contradictory evidence exists for both b enefi cial and detrimental eff ects of IL-6 aft er PNI, further investigation is warranted. and cytokine stimulation (Abe and Cavalli, 2008; O \200\231Brien and Nathanson, 2007). Micr oglia also respond to chemotactic factors and migrate towards the area in the spinal cord t hat receives the projections of degenerating C-fi bre primary aff erents (Beggs and Salter, 2007; Davalos et al., 2005; Nimmerjahn et al., 2005; Shehab et al., 2008; Shields et al., 2003). Interleukin-1 (IL-1 ) IL-1 is intimately associated with the initiation and mainte - nance of neuropathic pain (Gabay et al., 2011; Wolf et al., 2006). It is constitutively e xpressed in primary sensory neurons (Copray et al., 2001) and has pro-excitatory eff ects i n small and medium non-peptidergic neurons (Stemkowski and Smith, 2012) via a G protein-cou pled receptor mediated mechanism (von Banchet et al., 2011). PNI induces a rapid increase i n IL-1 in DRG macrophages (Th acker et al., 2007), sensitizing DRG neurons and contribut ing to increased mechanosensitivity (Ozaktay et al., 2006). Microglia express multiple receptors that may be involved in alter- ing reactivity (Beggs a nd Salter, 2010) that include: P2X and P2Y purinergic receptors (Trang et al., 2009; Tsuda et al., 2003), TLR-4 receptors (Ji and Strichartz, 2004; Lehnardt et al., 2003), and chem- okine receptors such as the fractalkine receptor CX3CR1 (Clark et al., 2011; Hughes et al., 2002), the CCR2 receptor for MCP-1/ CCL2 (Th acker et al., 2009), and the IFN receptor (Tsuda et al., 2009). Release of ATP, which acts via purinergic receptors, contrib- utes to microglia changing to a reactive phenotype (Haynes et al., 2006). Th is is accompanied by morphological changes, increased proliferative capacity, and increased expression of membra ne mol- ecules such as complement receptor 3 (CD11b; Kreutzberg, 1996; Nakajima and Kohsaka , 1998) and Iba1 (Beggs and Salter, 2010). Prostaglandins Prostaglandins, such as cyclo-oxygenase (COX)-2 dependent PGE 2 , have an im portant role in the pathogenesis of neuropathic pain (Ma and Quirion, 2008; Ma et al., 2010 ). At the spinal cord level, COX-2 is upregulated within the fi rst 3 days aft er injury (O \200\231Rielly and Loomis, 2006), and in the periphery, COX-2 and PGE 2 production is upre gulated in Schwann cells and macrophages infi ltrating dam- aged nerves. PGE 2 receptors, s uch as EP 1 receptors, are expressed on DRG neurons, and nerve injury causes the upregulati on of EP 1 in human and rat DRG neurons (Durrenberger et al., 2006; Ma et al., 2010). PGE 2 sensitizes damaged axons to mechanical and heat stimuli (Ma and Quirion, 2008; Michaelis e t al., 1998), and increases production of substance P and calcitonin gene-related peptide i n DRG neurons (Segond von Banchet et al., 2003). Pro-infl ammatory cytokines sensitize neurons by enhancing excitatory AMPA/NMDA currents an d reducing inhibitory gam- ma-aminobutyric acid (GABA)/glycine currents (Kawasaki et al., 2 008; Reeve et al., 2000; Sweitzer et al., 2001; Wang et al., 2005), but also contribute to neuropathic pain by stimulating microglia. Microglia are the main source of pro-infl ammato ry cytokines in the CNS (Hanisch, 2002) and increased functional microglial reactiv- ity in creases production and release of proinfl ammatory mediators (fractalkine IL-1 , IL-4, T NF ), prostaglandins, and ATP (Giaume et al., 2007; Kreutzberg, 1996; Segond von Banchet et al., 2003; Watkins et al., 2001). Following receptor activation, an increase in the int racellular concentration of Ca 2+ activates signaling path- ways, such as those mediated by the mitogen-activated protein kinase (MAPK) p38 (Ji and Suter, 2007) (Figure 7.1). p38 MAP K is a key factor in regulating the expression of proinfl ammatory cytokines, PGE2 and COX- 2 (Jana et al., 2003; Ji and Suter, 2007; Wilms et al., 2003), and is involved in the recru itment of microglia to areas of injury (Tsuda et al., 2005). p38 activation is increased fo llowing nerve damage, whereas its inhibition suppresses mechan- ical allodynia (Ji and Sute r, 2007; Jin et al., 2003; Svensson et al., 2003). Activation of the transcription factor N F-kB also promotes the production of proinfl ammatory cytokines, chemokines, COX-2 and othe r molecules related to immune activity (Y.-J. Gao and Ji, 2010). Nitric oxide (NO) Macrophages are a main source of nitric oxide (NO) aft er nerve injury (M oalem and Tracey, 2006), induced by TNF via induc- ible NO synthase (iNOS) (Lowenstein e t al., 1994). NO upregulates prostaglandin production and contributes to neuronal sensitiza tion (Mollace et al., 1997; Purwata, 2011). Nerve injury and spinal microglial reactivity Increased microglial reactivity has been demo nstrated in several animal models of PNI associated with chronic pain (Coull et al., 2005; Tsuda et al., 2003; Vega-Avelaira et al., 2009; Zhuang et al., 2005). Initiation of the mic roglial response aft er peripheral nerve damage is triggered by increased neural activity o f sensory aff er- ent fi bres (Suter et al., 2009). Nociceptive stimuli are predominantly t ransmitted by A - and C-fi bres (see Walker and Baccei, Chapter 6, this volume). However , following nerve injury, activity in A -fi bres which normally transduce innocuous tact ile sensation can contrib- ute to persistent pain (Matsumoto et al., 2008). Aff erent block ade with the local anesthetic bupivacaine, which blocks conduction in all sensory neurons ( C, A , and A fi bres) prevented nerve injury- induced spinal microglial activation; b ut resiniferatoxin, which selectively blocks peripheral input from C and A nociceptive f i bres (Mitchell et al., 2010) had no eff ect (Suter et al., 2009). Th is suggests that the large myelinated fi bres have an important role in the switch of microglia to a reactive p henotype aft er nerve injury. It has also been suggested that a small proportion of microgl ia are activity-independent, responding to retrograde axonal transportation IL-6 is upregulated in both the DRG and the spinal cord aft er nerve injury (Raghavendra et al., 2003), and also has a dual func- tion by stimulating microglia (Galiano et al., 2001; Klein et al., 1997) and neuronal responses to injury (Latr moli re et al., 2008). TLR4 receptor Th e metabotropic receptor TLR-4 is important for pathogen rec- ognition, and is activated by various substances, such as lipopoly- saccharide from Gram-negative bacter ia (H. Cao and Zhang, 2008; Capiralla et al., 2012). It is expressed on microglia and activ ation induces intracellular signaling cascades resulting in increased NF- B signaling an d subsequent expression of numerous genes regulating proinfl ammatory cytokines and other i mmunological molecules (Capiralla et al., 2012). Defi ciency of this receptor or its blocka de attenuates microglial reactivity, decreases levels of IFN , CHAPTER 7 neuroimmune interactions and pain during postnatal development 69 IL-1 , and TNF, and reduces pain behaviors associated with PNI (Tanga et al., 2005). the reduction in pain behaviors aft er nerve injury exhibited by the lymphocyte defi cient Rag1-null mice (Costigan et al., 2009). P2X 4 receptor P2X 4 receptors are expressed by microglia following PNI (Tsuda et al., 2003 ). Blockade of this receptor prevents and reverses mechanical allodynia in animal models of PNI, and mice defi - cient in P2X 4 do not develop mechanical hypersensitivity aft er nerv e injury (Ulmann et al., 2008). P2X 4 R expression is increased through the activation of o ther microglial receptors such us IFN and fi bronectin receptors (Masuda et al., 2012; T suda et al., 2008, 2009). P2X 4 R activation induces the synthesis and release of brain- de rived neurotrophic factor (BDNF) by microglia (Coull et al., 2005; Trang et al., 2009) via a p38 MAPK-dependent pathway (Beggs and Salter, 2010; Jin et al., 2003). BDNF activates its neuronal receptor tyrosine kinase B (TrkB) on lamina I neurons in the spinal cord dorsal h orn (Slack et al., 2005). Th is reduces the expression of the chloride transporter KCC2 lea ding to intracellular accumulation of Cl \210\222 . Th e net eff ect of this is disinhibit ion; reducing the inhibi- tory action of GABA, and facilitating further neuronal sensitizat ion (Coull et al., 2003, 2005). BDNF signaling in neurons also activates kinase pathways le ading to phosphorylation of the NMDA receptor NR1 subunit, which also contributes to enhanc ed sensitivity (Ren and Dubner, 2007; Suen et al., 1997). Postnatal di \200 erences in immune system activation after nerve injury Th e role of neur oimmune interactions as mediators of neuro- pathic pain has focused on experimental models in adult animals. However, clinical studies suggest that traumatic nerve injury is less lik ely to produce neuropathic pain in early life; brachial plexus avulsion, which causes inten se neuropathic pain in adults, is not painful when the injury is sustained at birth (Anand and Birch, 2002; see also Walker, Chapter 21, this volume). Laboratory stud- ies conducted in animals throughout postnatal development are consistent with this: in nerve injury model s of neuropathic pain, persistent mechanical allodynia does not develop if the injury is pe rformed in the fi rst 3 weeks of life, a developmental period which has parallels with huma n development from the neonate to adoles- cence (Howard et al., 2005; Ririe and Eisenach, 2 006). Since infant rats and humans clearly respond to acute noxious stimuli and dis- play b oth acute and chronic infl ammatory pain behavior from an early neonatal age, it appears th at the mechanisms underlying neu- ropathic pain are diff erentially regulated over a prolon ged postnatal period. Important diff erences include: \231 Microglia in the neonatal spinal cord are capable of being acti- vated by exogenous stimuli (e.g. intraspinal NMDA), but nerve injury in young rats fails to induce either the spinal microgliosis or macrophage accumulation in the DRG, that are hallmarks of PNI in the adult (Moss et al., 2007; Vega-Avelaira et al., 2007). CX3CR1 receptor Fractalkine (CX3CL1) is released by neurons and its receptor CX3CR1 is expr essed on microglia, making it a prime candi- date for neuronal-microglial signaling (Verge et al., 2004). Th e increased activity of primary aff erents aft er nerve injury releases f ractalkine into the extracellular space (Zhuang et al., 2007) and creates a chemotactic gra dient for microglia (Chapman et al., 2000). Intrathecal injection of fractalkine upregulate s expression of CX3CR1 and induces thermal hyperalgesia, which can be pre- vented by blocki ng fractalkine activity with CX3CR1 antibodies (Sun et al., 2007). Inhibiting microglial fu nction with minocycline also prevents the appearance of mechanical allodynia and thermal hy peralgesia induced by injection of fractalkine (Milligan et al., 2005). \231 Microarray analysis of gene expression in the DRG following nerve injury demonstrate changes associated with pro-infl am- matory cytokines such us IL-6, CSF-1, and MCP-1 being upregu- lated in adult but not in young rats (Vega-Avelaira et al., 2009). \231 In the spinal cord, many genes are diff erentially regulated in the adult compared w ith the infant rat, and the majority of these are involved in immune function (Costigan et al., 2009). \231 Activation and infi ltration of T-cells associated with the Th 1 pro-infl ammatory r esponse in the dorsal horn is much greater in adult rats than in young animals aft er nerve injury (Costigan et al., 2009). Lymphocyte in \201 ltration and central sensitization In addition to increased macrophage and microglial reactivity fol- lowing PNI, other immunocompetent cells, such as T-lymphocyt es also play a role. T lymphocytes can release excitatory cytokines, generating ectopic imp ulse activity in sensory neurons aft er nerve injury (L. Cao and DeLeo 2008; L. Cao et al., 2009; Costigan et al., 2009; Hu and McLachlan 2002). Th e dorsal root ganglia have a small population of lymphocytes which perform an immune sur- veillance function (Hu and McLachla n 2002). Peripheral nerve damage causes infi ltration of CD8+ lymphocytes into the DRG and spinal cord (Costigan et al., 2009; Hu and McLachlan 2002) which suggests that PNI triggers an adaptive immune response. Moreover, this lymphocyte infi ltration contributes to microg lia reactivity via CD40 (a member of the TNF-receptor superfamily) (L. Cao et al., 2009) an d mechanical hypersensitivity (Costigan et al., 2009; Hu and McLachlan 2002). Th e role of T cells is also demonstrated by Neonatal incision and long-term priming of microglial response Th e mechanisms and eff ecto rs previously described, that are in place to mute the postnatal immune response as part of the normal developmental process, would at fi rst suggest that early postnatal peripheral injury does not result in persistent neuropathic pain. While the incidence of neuropathic p ain following traumatic nerve injury is lower in infants and young children, the concept th at an absence of pain to early life injury indicates a lack of a response, whether CNS or i mmune, is simplistic. A key function that unites all sensory systems is that adult connecti vity is shaped by the bal- ance of sensory input in early postnatal life. In much the same way that auditory and visual system connectivity in the brain is driven by sound and light, tactile sense is driven by touch (Bourne, 2010; Sanes and Bao, 2009). Neural activity duri ng critical postnatal peri- ods can infl uence normal development (Hensch, 2004) and this i s SECTION 2 biological basis of pediatric pain 70 also true for nociceptive pathways (Beggs et al., 2002; Ren et al., 2004). A considerable c anon of evidence from both laboratory and clinical studies now shows that exposure to nocic eptive stimuli in early life can produce long-term changes in sensory processing and the re sponse to future injury (see Walker, Chapter 3; Grunau, Chapter 4, this volume). Importantl y, priming of neuroimmune signaling by neonatal injury can contribute to persistent eff ect s. \200\230Priming \200\231 or \200\230neonatal programming \200\231 of adult immune resp onses by early life immune stressors has been described (Boisse et al., 2004; Spencer et al ., 2011), and an early immune challenge can alter pain sensitivity in adulthood (Boisse et al., 2005). As microglia are long-lived cells and can retain an innate immune memory (Perry , 2010) they are well suited to a role in persistent alterations. Plantar hindpaw incision, an established model of postoperative pain, performed during the fi rst postnatal week, bu t not at older ages, enhances the response to subsequent injury 2 weeks later (Walker et al ., 2009). Th e impact of neonatal injury persists until adulthood, with increases in both t he degree and duration of incision-related hyperalgesia (Beggs et al., 2012a). Th ese funct ional changes are mirrored by alterations in the time course and degree of microglial react ivity in the dorsal horn. Th e primed state arises from centrally- mediated changes in dors al horn sensitivity or connectivity as: Beggs, S., Torsney C., Drew L. J., and Fitzgerald, M. (2002). Th e postnatal reorganization of primary aff erent input and dorsal horn cell receptive fi elds in the rat spinal cord i s an activity-dependent process. Eur J Neurosci , 16 (7), 1249 \200\2231258 . Beggs, S., Trang, T., and Salter, M. W. (2012b). P2X4R+ microglia drive neuropathic pain. Nat Neurosci, 15 , 1068 \200\2231073 . Boisse, L., Mouihate, A., Ellis, S., and Pittman, Q. J. (2004). Long-term alterations in ne uroimmune responses aft er neonatal exposure to lipopolysaccharide. J Neurosci, 24, 4928 \200\2234934 . 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 . Bourne, J. A. (2010). Unraveling the development of the visual cortex: implications for pla sticity and repair. J Anat , 217 (4), 449 \200\223468 . doi:10.1111/j.1469-7580.2010.01275. x. Br zda, V., Klus kov , I., Sv zensk , I., Veselkov , Z., and Dubov , P . (2009). Bilateral changes in IL-6 protein, but not in its receptor Gp130, in rat dorsal r oot ganglia following sciatic nerve ligature. Cell Mol Neurobiol , 29 (6 \200\2237), 1053 \200\2231062 . doi:10.1007/s10571-009-9396 \200\2230. Cao, H. and Zhang, Y.-Q. (2008). Spinal glial activation contributes to pathological pain s tates. Neurosci Biobehav Rev , 32 (5), 972 \200\223983 . doi:10.1016/j.neubiorev.2008.03.00 9. Cao, L. and DeLeo, J. A. (2008). CNS-infi ltrating CD4+ T lymphocytes contribute to murine spinal nerve transection-induced neuropathic pain. Eur J Immunol , 38 (2), 448 \200\223458 . doi:10.1002/eji.200737485. \231 Hyperalgesia is selectively reversed by intrathecal minocycline, Cao, L., Palmer, C. D., Malon J. T., and DeLeo, J. A. (2009). Critical role of microglial C D40 in the maintenance of mechanical hypersensitivity in a murine model of neuropathic pain . Eur J Immunol , 39 (12), 3562 \200\2233569 . doi:10.1002/eji.200939657. which reduces microglial reactivity in the spinal cord. \231 Th e increased sensitivity and microglial reactivity following neo- natal incision i s also seen following direct electrical stimulation of the tibial nerve, which bypasses per ipheral nociceptors (Beggs et al., 2012a). Capiralla, H., Vingtdeux, V., Zhao, H., Sankowski, R., Al-Abed, Y., Davies, P., et al . (20 12). Resveratrol mitigates lipopolysaccharide- and A - mediated microglial infl ammation by inhibiting the TLR4/NF- B/STAT signaling cascade. J Neurochem , 120 (3), 461 \200 \223472 . doi:10.1111/j.1471- 4159.2011.07594.x. Conclusion Interactions between the immune system and nociceptive pathways play an importan t role in modulating pain sensitivity. Th roughout postnatal development, neuroimmune inter actions have a signifi - cant impact on age-related changes in the response to PNI, and als o play a role in persistent changes in future pain response following neonatal surgical inj ury. Evaluating the relative risks and benefi ts of modulating microglial activity to preve nt long-term changes in nociceptive pathways, or to manage enhanced pain sensitivity states , requires further research. Chang, Y.-W. and Waxman S. G. (2010). Minocycline attenuates mechanical allodynia and centr al sensitization following peripheral second-degree burn injury. J Pain, 11 (11), 1146 \200 \2231154 . doi:10.1016/j.jpain.2010.02.010. Chapman, G. A., Moores, K., Harrison, D., Campb ell, C.A., Stewart, B. R., and Strijbos, P. J. (2000). Fractalkine cleavage from neuronal m embranes represents an acute event in the infl ammatory response to excitotoxic brain damag e. J Neurosci , 20 (15), RC87 . Chiang, C. Y., Sessle, B. J., and Dostrovsky, J. O. (2012). Role of astrocytes in pain. Neurochem Res, 37 , 2419 \200\2232431 . Chiu, I. M., Von Hehn, C. A., and Woolf, C. J. (2012). Neurogenic infl ammation and the per ipheral nervous system in host defense and immunopathology. Nat Neurosci, 15 , 1063 \200 \2231067 . Clark, A. K., Staniland, A. A., and Malcangio, M. (2011). Fractalkine/CX3CR1 signaling in c hronic pain and infl ammation. Curr Pharm Biotechnol , 12 (10), 1707 \200\2231714 . References Abbadie, C., Lindia, J. A., Cumiskey, A. M., Peterson, L. B., Mudgett, J. S., Ba yne, E. K., et al . (2003). Impaired neuropathic pain responses in mice lacking the chemoki ne receptor CCR2. Proc Natl Acad Sci U S A , 100 (13), 7947 \200\2237952 . doi:10.1073/pnas .1331358100. Copray, J. C., Mantingh, I., Brouwer, N., Biber, K., K st, B. M., Liem, R. S., et al . ( 2001). Expression of interleukin-1 beta in rat dorsal root ganglia. Curr Pharm Biotechnol , 118 (2), 203 \200\223211 . Abe, N. and Cavalli, V. (2008). Nerve injury signaling. Curr Opin Neurobiol , Costigan, M., Moss, A., Latremoliere, A., Johnston, C., Verma-Gandhu, M., Herbert T. A., et al . (2009). T-cell infi ltration and signaling in the adult dorsal spinal cord is a major contributor to neuropathic pain- like hypersensitivity. J Neurosci , 29 (46), 14415 \200 \22314422 . doi:10.1523/ JNEUROSCI.4569-09.2009. 18 (3), 276 \200\223283 . doi:10.1016/j.conb.2008.06.005. Anand, P. and Birch, R. (2002). Restoration of sensory function and lack of long-term chron ic pain syndromes aft er brachial plexus injury in human neonates. Brain , 125 (Pt 1), 113 \200\223122 . Beggs, S., Currie, G., Salter, M. W., Fitzgerald, M., and Walker, S. M. (2012a). Priming of adult pain responses by neonatal pain experience: maintenance by central neuroimmune activ ity. Brain, 135 , 404 \200\223417. Beggs, S. and Salter, M. W. (2007). Stereological and so matotopic analysis of the spinal microglial response to peripheral nerve injury. Brain Beha v Immun , 21 (5), 624 \200\223633 . doi:10.1016/j.bbi.2006.10.017. Costigan, M., Scholz, J., and Woolf, C. J. (2009). Neuropathic pain: a maladaptive response of the nervous system to damage. Annu Rev Neurosci , 32 , 1 \200\22332 . doi:10.1146/annur ev.neuro.051508.135531. Coull, J. A. M., Beggs, S., Boudreau, D., Boivin D., Tsuda, M., Inoue, K., et al . (2005). BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain . Nature , 438(7070), 1017 \200\2231021 . doi:10.1038/nature04223. Beggs, S. and Salter M. W. (2010). Microglia-neuronal signaling in neuropathic pain hyperse nsitivity 2.0. Curr Opin Neurobiol , 20 (4), 474 \200\223 480 . doi:10.1016/j.conb.2010.08. 005. Coull, J. A. M., Boudreau, D., Bachand, K., Prescott, S. A, Nault, F., S k, A., et al . (2003). Trans-synaptic shift in anion gradient in spinal lamina I CHAPTER 7 neuroimmune interactions and pain during postnatal development 71 Hu P., Bembrick, A. L., Keay, K. A., and McLachlan, E. M. (2007). Immune cell involvement i n dorsal root ganglia and spinal cord aft er chronic constriction or transection of the rat sciatic nerve. Brain Behav Immun , 21 (5), 599 \200\223616. neurons as a mechanism of neuropathic pain. Nature , 424 (6951), 938 \200\223 942 . doi:10. 1038/nature01868. Davalos, D., Grutzendler, J., Yang, G., Kim, J. V., Zuo, Y., Jung, S., et al . (2005). ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci , 8 (6), 752 \200\223758 . doi:10.1038/nn1472. Hu, P. and McLachlan, E. M. (2002). Macrophage and lymphocyte invasion of dorsal root gangl ia aft er peripheral nerve lesions in the rat. Neuroscience , 112 (1), 23 \200\22338 . DeLeo, J. A., Colburn, R. W., Nichols, M., and Malhotra, A. (1996). Interleukin-6-mediated hyperalgesia/allodynia and increased spinal IL-6 expression in a rat mononeuropathy model. J Interferon Cytokine Res , 16 (9), 695 \200\223700 . Hu, P. and McLachlan, E. M. (2003). Distinct functional types of macrophage in dorsal root ganglia and spinal nerves proximal to sciatic and spinal nerve transections in the rat. Exp Neurol , 184 (2), 590 \200\223605 . doi:10.1016/ S0014 \200\2234886(03)00307 \200\2238. Durrenberger, P. F., Facer, P., Casula, M. A., Yiangou, Y., Gray, R. A., Chessell, I. P., e t al . (2006). Prostanoid receptor EP1 and Cox-2 in injured human nerves and a rat model of nerve injury: a time-course study. BMC Neurol , 6 , 1. doi:10.1186/1471-2377-6-1. Hughes, P. M., Botham, M. S., Frentzel, S., Mir, A., and Perry, V. H. (2002). Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, during acute and chronic infl ammation in t he rodent CNS. Glia , 37 (4), 314 \200\223327 . Fadel, S. and Sarzotti, M. (2000). Cellular immune responses in neonates. Int Rev Immunol , 19 ( 2 \200\2233 ), 173 \200\223193 . Jana, M., Dasgupta, S., Saha, R. N., Liu, X., and Pahan, K. (2003). Induction of tumor necr osis factor-alpha (TNF-alpha) by interleukin-12 P40 monomer and homodimer in microglia and macrophages. J Neurochem , 86 (2), 519 \200\223528 . Fitzgerald, M. (2005). Th e development of nociceptive circuits. Nat Rev Neurosci , 6(7), 507 \200\223520. doi:10.1038/nrn1701. Fitzgerald, M., Butcher, T., and Shortland, P. (1994). Developmental changes in the laminar termination of a fi bre cutaneous sensory aff erents in the rat spinal cord dorsal horn. J Comp Neurol , 348 (2), 225 \200\223233 . doi:10.1002/ cne.903480205. Jeon, S.-M., Sung, J.-K., and Cho, H.-J. (2011). Expression of monocyte chemoattractant pro tein-1 and its induction by tumor necrosis factor receptor 1 in sensory neurons in the vent ral rhizotomy model of neuropathic pain. Neuroscience , 190 , 354 \200\223366 . doi:10.1016 /j. neuroscience.2011.06.036. Gabay, E., Wolf, G., Shavit, Y., Yirmiya, R., and Tal, M. (2011). Chronic blockade of inter leukin-1 (IL-1) prevents and attenuates neuropathic pain behavior and spontaneous ectopic n euronal activity following nerve injury. Eur J Pain , 15 (3), 242 \200\223248 . doi:10.1016 /j.ejpain.2010.07.012. Galiano, M., Liu Z. Q., Kalla, R., Bohatschek, M., Koppius, A., Gsch wendtner, A., et al . (2001). Interleukin-6 (IL6) and cellular response to facial nerve inj ury: eff ects on lymphocyte recruitment, early microglial activation and axonal outgrowth i n IL6-defi cient mice. Eur J Neurosci , 14 (2), 327 \200\223341 . Gao, X., Kim, H. K., Chun g, J. M., and Chung, K. (2005). Enhancement of NMDA receptor phosphorylation of the spinal dorsal horn and nucleus gracilis neurons in neuropathic rats. Pain , 116 ( 1 \200\2232 ), 6 2 \200\22372 . doi:10.1016/j. pain.2005.03.045. Ji, R.-R., Gereau 4th, R. W., Malcangio, M., and Strichartz, G. R. (2009). MAP kinase and p ain. Brain Res Rev , 60 (1), 135 \200\223148 . doi:10.1016/j. brainresrev.2008.12.011. Ji, R.-R. and Strichartz, G. (2004). Cell signaling and the genesis of neuropathic pain. Sci STKE , 2004 (252), reE14. doi:10.1126/stke.2522004re14. Ji, R.-R. and Suter, M. R. (2007). P38 MAPK, microglial signaling, and neuropathic pain. Mol Pain , 3 , 33. doi:10.1186/1744-8069-3-33. Jin, S.-X., Zhuang, Z.-Y., Woolf, C. J., and Ji, R.-R. (2003). P38 mitogen- activated prote in kinase is activated aft er a spinal nerve ligation in spinal cord microglia and dorsal r oot ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci , 23 (10), 4017 \200\2234022 .