Justificación de la actividad investigadora

Sensory signals are processed in the spinal dorsal horn by the convergence of excitatory input from afferent fibers, local excitation and inhibition from interneurons, and descending supraspinal inhibition (Fig. 1.3). Nociception can, therefore, be augmented or suppressed by changes in those convergent inputs that alter dorsal horn neuronal excitability. Central sensitization is a state in which nociceptive signaling is amplified by spinal neuronal hyperexcitability, resulting in persistent pain that outlasts the inciting injury or tissue damage (Latremoliere and Woolf, 2009). Central sensitization is characterized by altered neuronal function in the spinal cord that includes increased spontaneous activity, reduced thresholds for activation, heightened responses to both non- noxious and noxious stimuli, and expansion of receptive fields (Coderre et al., 1993; Latremoliere and Woolf, 2009; Woolf, 1983; Woolf and Salter, 2000). Those functional changes in the central nervous system (CNS) result in spontaneous pain, painful responses to typically non-noxious stimuli like light touch (allodynia), and hypersensitivity to noxious stimuli at the site of injury (primary hyperalgesia) and at regions distant from the site of injury (secondary hyperalgesia) (Coderre et al., 1993; Lamotte et al., 1991).

Central sensitization is initiated by sustained afferent firing that occurs during noxious stimulation or tissue injury (Seltzer et al., 1991a; Wall et al., 1974). Blocking the increased afferent discharge that accompanies nerve injury effectively reduces the subsequent development of central sensitization in models of neuropathic pain (Dougherty et al., 1992; Gonzales-Darder et al., 1986; Seltzer et al., 1991a). Blocking injury-induced afferent discharge is also a foundational concept for the use of preemptive

analgesia in order to reduce postoperative pain (Coderre et al., 1993; Woolf and Chong, 1983; Woolf and Wall, 1986). Once spinal neuronal hyperexcitability develops, neurons can return to a baseline state in the absence of continuing afferent discharge, but hyperexcitability can be maintained by low levels of firing, such as the ectopic discharge that develops in damaged neural tissue (Devor, 2009; Devor et al., 1992; Djouhri et al., 2006; Koltzenburg et al., 1992; Xie et al., 2005). Although excessive stretch of the facet capsule has been shown to induce increased firing of the afferents that innervate the joint (Chen et al., 2006; Lu et al., 2005a), no study has evaluated the role of injury-induced afferent discharge from the joint in the development and maintenance of persistent facet- mediated pain.

Many of the changes in the spinal dorsal horn that are associated with central sensitization involve altered glutamatergic signaling. Glutamate is the primary excitatory neurotransmitter in the CNS (Sheng and Lin, 2001; Yaksh, 2006). Glutamate activates several different ionotropic and metabotropic receptors on the post-synaptic membrane (Fig. 1.5a). Ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors

(AMPAR) are permeable to Na+ and K+ currents and mediate rapid excitatory

neurotransmission (Yaksh, 2006). N-methyl-D-aspartate receptors (NMDARs) are a second class of ionotropic receptor that are activated by glutamate in a voltage-dependent manner and, once activated, allow calcium influx into the postsynaptic terminal (Fig. 1.5b) (Petrenko et al., 2003; Sheng and Lin, 2002; Yaksh 2006). Metabotropic glutamate receptors (mGluRs) are a family of G protein-coupled receptors that mediate slower synaptic responses. Glutamate binding to group 1 metabotropic receptors, namely

mGluR1 and mGluR5, can also contribute to calcium influx by releasing calcium from intracellular stores (Fig. 1.5b) (Sheng and Lin, 2002; Yaksh, 2006).

Central sensitization is induced by increases in intracellular calcium from NMDAR and group 1 mGluR activation during sustained afferent discharge in the dorsal horn (Fig. 1.5) (Petrenko et al., 2003; Soliman et al., 2002; Woolf and Thompson, 1991; Young et al., 1997). Calcium influx activates calcium-dependent kinases, including PKC, PKA, CamKII, and ERK, that act on numerous targets to directly influence excitatory signaling (Fig. 1.5c) (Kawasaki et al., 2004; Latremoliere and Woolf, 2009). For example, phosphorylation of AMPARs and NMDARs at multiple sites increases their activation kinetics and trafficking to the membrane, enhancing membrane excitability

Fig. 1.5. The initiation of central sensitization in the dorsal horn. (a) Glutamatergic

signaling activates ionotropic (AMPAR, NMDAR) and metabotropic (mGluR5)

receptors. (b) NMDAR and mGluR5 activation cause calcium influx from external

and intracellular sources like the endoplasmic reticulum (ER). (c) Calcium influx

activates kinases (PKC, ERK, and CamKII) and voltage-dependent calcium channels (VDCC) to increase membrane excitability and the strength of excitatory synapses, leading to neuronal hyperexcitability (adapted from Latremoliere and Woolf, 2009).

mGluR5 _NMDAR AMPAR

VDCC

mGluR5 _NMDAR AMPAR

VDCC

mGluR5 _NMDAR AMPAR

VDCC PKC ERK CaMKII Ca2+ _glutamate a) b) c) ER ER ER

(Daulhac et al., 2011; Liu and Salter, 2010; Petrenko et al., 2003; Ultenius et al., 2006). Activation of calcium-dependent kinases also leads to transcriptional changes that alter expression of proteins in primary afferent and dorsal horn neurons to further enhance spinal excitability (Kawasaki et al., 2004; Latremoliere and Woolf, 2009).

Temporal and spatial management of glutamate concentrations in synapses is important for preventing aberrant signaling and excitotoxicity. Glutamate signaling is, therefore, regulated by astrocytic and neuronal transporters that remove glutamate from the synapse (Danbolt, 2001; Liaw et al., 2005). The excitatory amino acid transporters (EAAT1-5) are one family of glutamate transporters that are crucial for the regulation of extracellular glutamate concentrations in the spinal dorsal horn (Queen et al., 2007). EAAT1 and EAAT2 are homologous to two glutamate transporters in the rat, glutamate aspartate transporter (GLAST) and glial glutamate transporter 1 (GLT1), and are expressed primarily on astrocytes. A third member of the transporter family, EAAT3, is neuronally expressed and is homologous to the rat glutamate transporter EAAC1 (Queen et al., 2007). In rodent models of neuropathic pain, downregulation of GLAST and GLT1 contributes to persistent behavioral sensitivity (Hu et al., 2009; Sung et al., 2003; Xin et al., 2009), but dorsal horn expression of those astrocytic transporters has not been evaluated in the context of facet joint-mediated pain.

Astrocytes can also contribute to central sensitization and pathological pain after tissue injury. Astrocytes can be activated by excitatory signaling molecules, including substance P, glutamate, and ATP, that are released by primary afferent nociceptors in response to tissue damage or noxious stimulation (Milligan and Watkins, 2009). Once activated, astrocytes increase their expression of glial fibrillary acidic protein (GFAP),

vimentin, and nestin, which are intermediate filament proteins that form part of the intracellular cytoskeletal network, resulting in hypertrophy of cellular processes (Benveniste, 1992; Pekny and Nilsson, 2005). Activated astrocytes in the dorsal horn also release substances that can both enhance neuronal excitability (i.e., excitatory neurotransmitters, growth factors, and prostaglandins) and promote spinal inflammation (i.e., pro-inflammatory cytokines) (Benveniste, 1992; Watkins et al., 2001). Astrocytes may play a particularly important role in facet joint-mediated pain, because spinal GFAP is upregulated in conjunction with the development of persistent behavioral sensitivity after injurious facet joint loading (Lee et al., 2004a; Lee et al., 2008). Separately, anti- inflammatory treatments and spinal treatments that attenuate neuronal excitability reduce GFAP expression in parallel with attenuation of behavioral sensitivity, further supporting the contribution of astrocyte activation to facet-mediated pain (Dong et al., 2013a; Dong et al., 2013b).