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In addition, it is increasingly recognized that interactions between immune cells and nociceptive pathways can modulate pain sensitivity. Th e role and function of immune cells in the central nervous system changes during postnatal development , and as a result, the impact of neuroimmune interactions on pain signaling varies with bot h age and the type of injury. Peripheral nerve injury Neuropathic pain can be severe and diffi cult to treat (see also Wa lker, Chapter 21; Rastogi and Campbell, Chapter 49, this volume). Despite considerable eff ort the conditions that initiate and maintain neuropathic pain are as yet not fully underst ood. Peripheral nerve injury (PNI) activates multiple cellular and molecular pathways in bo th the peripheral and central nervous systems. Of consider- able interest now is parsing th ese cellular and molecular pathways; PNI will evoke a nerve regeneration response and a str ess response associated with interrupted metabolic pathways (Costigan et al., 2009). Clearl y, explicit damage to the peripheral nervous system is countered by responses in multiple s ystems, and it is the interaction between immune system reactivity and injury-driven change s in nociceptive signaling that is now under scrutiny. Introduction Interactions between nociceptive pathways and the immune sys- tem are importan t for detecting and responding to potential harm from tissue damage and infection, and invo lve multiple diff erent cell types in diff erent regions (Figure 7.1 ). In the periphery, n oci- ceptors release a range of mediators which not only contribute to peripheral sensitiza tion, but also attract and activate immune cells (mast cells, dendritic cells, T lymphocyte s) to facilitate removal of pathogens and wound healing (Chiu et al., 2012). Macrophages ar 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).