Are the emergence of affective disturbances in neuropathic pain states contingent on supraspinal neuroinflammation?

Accepted Manuscript Are the emergence of affective disturbances in neuropathic pain states contingent on supraspinal neuroinflammation? Nathan T. Fiore, Paul J. Austin PII: DOI: Reference:

S0889-1591(16)30103-9 http://dx.doi.org/10.1016/j.bbi.2016.04.012 YBRBI 2859

To appear in:

Brain, Behavior, and Immunity

Received Date: Revised Date: Accepted Date:

20 January 2016 11 April 2016 22 April 2016

Please cite this article as: Fiore, N.T., Austin, P.J., Are the emergence of affective disturbances in neuropathic pain states contingent on supraspinal neuroinflammation?, Brain, Behavior, and Immunity (2016), doi: http://dx.doi.org/ 10.1016/j.bbi.2016.04.012

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Review article: Are the emergence of affective disturbances in neuropathic pain states contingent on supraspinal neuroinflammation?

Nathan T. Fiore, Paul J. Austin

Discipline of Anatomy & Histology, School of Medical Sciences, The University of Sydney, Sydney, NSW 2006, Australia

Corresponding author: Paul J. Austin

Laboratory of Neuroimmunology and Behaviour, Room E513, Anderson Stuart Building (F13), University of Sydney, NSW, 2006, Australia Email: [email protected] Telephone: +61 (0) 293515061

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Abstract Neuro-immune interactions contribute to the pathogenesis of neuropathic pain due to peripheral nerve injury. A large body of preclinical evidence supports the idea that the immune system acts to modulate the sensory symptoms of neuropathy at both peripheral and central nervous system sites. The potential involvement of neuroimmune interactions in the highly debilitating affective disturbances of neuropathic pain, such as depression, anhedonia, impaired cognition and reduced motivation has received little attention. This is surprising given the widely accepted view that sickness behaviour, depression, cognitive impairment and other neuropsychiatric conditions can arise from inflammatory mechanisms. Moreover, there is also a set of well-described immune-to-brain transmission mechanisms that explain how peripheral inflammation can lead to supraspinal neuroinflammation. In the last 5 years increasing evidence has emerged that peripheral nerve injury induces supraspinal changes in cytokine or chemokine expression and alters glial cell activity. In this systematic review, based on strong preclinical evidence, we advance the argument that the emergence of affective disturbances in neuropathic pain states are contingent on pro-inflammatory mediators in the interconnected hippocampal-medial prefrontal circuitry that subserve affective behaviours. We explore how dysregulation of inflammatory mediators in these networks may result in affective disturbances through a wide variety of neuromodulatory mechanisms. There are also promising early findings that anti-inflammatory agents have efficacy to treat a variety of neuropsychiatric conditions including depression and appear suited to sub-groups of patients with elevated pro-inflammatory profiles. Thus, although further research is required, aggressively targeting supraspinal pro-inflammatory mediators at critical

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time-points in appropriate clinical populations is likely to be a novel avenue to treat debilitating affective disturbances in neuropathic conditions.

Keywords Neuropathic pain, cytokines, astrocytes, microglia, neuroinflammation, hippocampus, medial prefrontal cortex, affective behaviour

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1. Introduction Neuropathic pain is defined as ‘pain arising as a direct consequence of a lesion or disease affecting the somatosensory system’ (Treede et al., 2008), most commonly due to traumatic or iatrogenic injury, diabetes or infection. Clinically, neuropathic pain is characterised by both sensory and affective disturbances. Sensory disturbances include; spontaneous pain, allodynia, hyperalgesia, dysaesthesia and paraesthesia. The highly debilitating affective disturbances described by patients include impaired cognition, working memory dysfunction, decreased appetite, anhedonia, depression, disruption to sleep-wake cycles, as well as disturbances in familial and social interactions (Apkarian et al., 2004; Backonja et al., 2008; Bertolucci and de Oliveira, 2013; Dick and Rashiq, 2007; Fishbain et al., 1997; Hart et al., 2000; Jensen and Finnerup, 2007; Menefee et al., 2000; Meyer-Rosberg et al., 2001; Moriarty et al., 2011; Samwel et al., 2006). It should also be noted that the degree of disability associated with affective disturbances varies greatly between individuals, even in the case of post-surgical neuropathic pain cohorts where causality is less varied (Kehlet et al., 2006).

In the last 25 years researchers have developed four widely used preclinical nerve injury models in order to replicate the symptoms of neuropathic pain. They involve damaging a portion of axons which contribute to the sciatic nerve, and from greatest to least neuronal damage include: spinal nerve ligation (SNL), where the L5 and/or L6 spinal nerves are ligated (Kim and Chung, 1992); spared nerve injury (SNI), where the tibial and common peroneal sciatic nerve branches are tightly ligated then transected (Decosterd and Woolf, 2000); partial sciatic nerve ligation (PSNL), where approximately half of the sciatic nerve is ligated (Seltzer et al., 1990); and chronic

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constriction injury (CCI), where four ligatures are loosely tied around the sciatic nerve (Bennett and Xie, 1988). In general these peripheral nerve injury models have similar time courses of sensory symptoms (emerging within 24 hours and persisting >2months), though signs of allodynia are greatest following SNL and smallest after CCI, and the most prominent behavioural signs of ongoing pain occur after CCI (for review see Kim et al., 1997). Further, the SNI model uniquely fails to produce thermal hyperalgesia, this may be explained by a lack of denervated Schwann cells which are known to produce many neuroactive molecules capable of acting on intact axons (for review see Costigan et al., 2009b; Decosterd and Woolf, 2000). Expression of affective disturbances, including anxiety- and depressive-like behaviours, sleep disturbances, anhedonia and changes in social interactions have been observed following all four nerve injury models, emerging approximately 1-2 weeks after injury, although there are also a number of studies which reported no changes in these behaviours (for review see Liu and Chen, 2014).

Nerve injury models, in combination with evoked-reflexive responses, have established that sensory disturbances, such as allodynia and hyperalgesia are a consequence of interactions between neurons, inflammatory immune and immunelike glial cells, as well as various immune-cell derived inflammatory mediators, in particular cytokines and chemokines, but also ATP, histamine, bradykinin and prostaglandins (PGs) (for review see Austin and Moalem-Taylor, 2010, 2013; Grace et al., 2014; Martini and Willison, 2016). Thus establishing neuropathic pain as a neuro-immune disorder. Nerve injury induces the release of inflammatory mediators at multiple levels of the neuraxis; strongest at the site of injury where it facilitates Wallerian degeneration and recovery, but also in the dorsal root ganglia (DRG) and

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spinal cord recipient segments, and it is these peripheral and spinal sites of inflammation that appear to contribute to the development and maintenance of allodynia and hyperalgesia.

Peripheral neuropathy also induces production of inflammatory mediators within the brain, and this review will focus predominantly on preclinical findings of nerve injury induced alterations in expression of pro-inflammatory cytokines and glial cell activation markers. Although other inflammatory and chemical mediators have been implicated in pain neurobiology, changes in their expression patterns in the neuropathic brain have yet to be described. We will only briefly mention human findings mainly because brain-imaging technology is only just making it possible to investigate glial activation (Alshelh et al., 2016; Loggia et al., 2015), and analysis of cytokine expression profiles has only been examined through skin biopsies, or the collection of blood and CSF from chronic pain patients.

Surprisingly little attention has been paid to the potential role of supraspinal neuroinflammation in the emergence of affective disturbances in neuropathic pain states, which can be hypothesised on the basis of our understanding of the immune mechanisms of sickness behaviour, as well as evidence that cognitive impairment, depression and other neuropsychiatric disorders are associated with pro-inflammatory cytokines and chemokines (Capuron et al., 2002; Capuron and Miller, 2011; Capuron et al., 2005; Dantzer, 2009; Dantzer et al., 2008; Dowlati et al., 2010; McAfoose and Baune, 2009; Stuart and Baune, 2014).

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In this review, we advance the argument that the emergence of affective disturbances in neuropathic pain states are contingent on altered inflammatory mediators in the interconnected hippocampal-medial prefrontal circuitry that subserves cognitive, emotional and motivational behaviours (i.e. affective behaviours). In our critical evaluation of the literature we find growing evidence that nerve injury evokes anatomically specific neuroinflammation in affective forebrain regions that is causally responsible for affective disturbances (Burke et al., 2013a; Dellarole et al., 2014; Mor et al., 2010; Narita et al., 2006; Nascimento et al., 2015; Norman et al., 2010b; Ren et al., 2011; Taylor et al., 2015; Wang et al., 2013a; Wu et al., 2014). Given the neural circuits mediating motivational, emotional and cognitive behaviours are highly conserved across species, we argue observations in these preclinical models are likely translatable to the human neuropathic pain experience (Craig, 2003; Keay et al., 2004; Murray et al., 2011). Thus the aim of this review is to systematically present the preclinical evidence that nerve-injury evoked supraspinal neuroinflammation underlies the development of affective disturbances by disrupting critical physiological processes. Following presentation of the preclinical evidence, we will discuss the clinical potential of anti-inflammatory treatments for neuropathic pain and other neuropsychiatric conditions.

2. Peripheral inflammation and affective disturbances It is well established that peripheral inflammation or an immune challenge in animals and humans triggers a set affective disturbances termed sickness behaviour (for review see Dantzer et al., 2008). Moreover, peripheral immune challenge appears to underlie modified behaviours through an anatomically specific pattern of supraspinal neuroinflammation, given both are antagonised by intracerebroventricular (i.c.v.)

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injection of the anti-inflammatory cytokine interleukin-10 (IL-10) or minocycline treatment (Ban et al., 1992; Biesmans et al., 2013; Bluthe et al., 1999; Castanon et al., 2004; Gabellec et al., 1995; Henry et al., 2008; Laye et al., 2000; Laye et al., 1994; Siren et al., 2001; Utsuyama and Hirokawa, 2002).

Clinically, major depression disorder has a strong inflammatory component, with elevated blood levels of the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) in depressed individuals (Dowlati et al., 2010; Liu et al., 2012), and interferon-α (IFNα) immunotherapy is known to cause widespread neuropsychiatric symptoms (Capuron et al., 2002; Capuron et al., 2007; Pavol et al., 1995). Further, patients with painful neuropathy coupled with depression have been observed to have higher TNF levels than painful neuropathy patients without depression (Uceyler et al., 2007), an observation which supports the idea that comorbid symptoms of pain and depression may share a common inflammatory mechanism (Walker et al., 2014). Taking this idea a step further, we will now present evidence that an individual’s inflammatory response to injury or disease may predispose them to persistent pain and affective disturbances.

Keay and colleagues have developed a preclinical model to explore individual differences in neuropathic pain and disability using CCI combined with residentintruder social interactions testing and sleep-wake analysis in rats (Keay et al., 2004; Monassi et al., 2003). The major finding from these studies was that CCI leads to a persistent reduction in dominance behaviour and disrupted sleep-wake cycles in only a sub-group of rats, despite all nerve-injured rats having equivalent levels of allodynia. In a recent study utilising this model, we found that the sub-group of rats

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with persistent disability in social interactions had significantly increased expression of T lymphocytes in the injured sciatic nerve compared to rats that displayed no affective disturbances (Austin et al., 2015). Within this disabled sub-group: IL-6 and the chemokine monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) in the injured sciatic nerve, IL-6 in the DRG, and interleukin-1β (IL-1β) in the spinal cord were all selectively increased (Austin et al., 2015). Thus a distinct cytokine signature related to the enhanced adaptive immune response may underlie affective disturbances in this sub-group of nerve-injured rats.

3. Immune-to-brain signalling and inflammatory cytokines as neuromodulators Release of inflammatory cytokines may occur in response to the local environment in the central nervous system, but also due to the release of peripheral cytokines triggered by a distant inflammatory event (Mitchell et al., 2009). In the context of peripheral nerve injury, induction of MCP-1 and IL-1β compromise the integrity of the blood-spinal cord barrier increasing its permeability (Echeverry et al., 2011), which allows spinal cord infiltration of circulating T cells and monocytes (Cao and DeLeo, 2008; Costigan et al., 2009a; Fleming et al., 2009). In a more general context, the current understanding of immune-to-brain signalling is that three non-exclusive pathways exist; a) neural transmission, b) humoral transmission and c) molecular transmission (see Figure 1). We direct readers to the excellent recent review by Banks (2015) on the transmission of inflammatory mediators across the blood-brain barrier. When attempting to integrate the neural transmission route into the neuropathic setting, it should be noted that this could apply to an injured peripheral nerve, such as the sciatic nerve. Transmission would be via primary afferent fibres projecting to

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dorsal horn neurons of the ascending anterolateral pathways, which in turn project to numerous supraspinal regions.

An emerging immune-to-brain transmission route could involve communication between the recently described ‘glymphatic system’ which enables passive circulation of interstitial fluid between the brain parenchyma and the subarachnoid CSF along paravascular spaces (Iliff et al., 2012; Yang et al., 2013), and the meningeal lymphatic vessels that carry leukocytes and drain into both deep and superficial cervical lymph nodes (Louveau et al., 2015).

Supraspinal cytokines and chemokines act in a neuromodulatory fashion, sharing similarities with neuropeptides, and operate under normal physiological conditions within a ‘cytokine network’ of neurons, microglia and astrocytes that are able to regulate cytokine production, express cytokine receptors and both amplify and attenuate cytokine signals (Haas and Schauenstein, 1997; Rothwell et al., 1996; Vitkovic et al., 2000). The magnitude of nerve-injury evoked supraspinal neuroinflammation is relatively mild compared to the peripheral site of nerve injury, or classical ‘neuroinflammation’ associated with an ischemic event, cell death or neurodegenerative disorders. Nevertheless, it appears to cause a perturbation in physiological cytokine concentrations sufficient to disrupt normal neuronal functioning, in what has been described as a ‘sub-inflammatory’ response (Adler et al., 2005; Adler and Rogers, 2005; Hutchinson and Watkins, 2014). A non-exhaustive summary of possible neuromodulatory mechanisms by which a dysregulation of inflammatory cytokines may underlie the development of affective behavioural changes is outlined in Figure 2. It is important to appreciate that the action of

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inflammatory cytokines throughout the brain occurs in an anatomically specific fashion as cytokine and chemokine receptor expression is not ubiquitous, with regions critical for affective state regulation showing particularly strong expression patterns (i.e., hippocampus, hypothalamus, amygdala, nucleus accumbens and cortical regions) (Ban et al., 1991; Banisadr et al., 2002a; Banisadr et al., 2002b; Farrar et al., 1987; Gadient and Otten, 1993, 1994; Horuk et al., 1997; Kinouchi et al., 1991; Schöbitz et al., 1993; Takao et al., 1990; van der Meer et al., 2000; Yan et al., 1992).

4. Inflammatory cytokines and affective disturbances in the neuropathic brain Distinct anatomical regions of the hippocampus and mPFC, as well as interconnected regions such as the striatum/NAcc, ventral tegmental area (VTA), hypothalamus, amygdala and periaqueductal gray (PAG) are critical in regulating affective behaviours. Peripheral neuropathy produces elevated expression of pro-inflammatory cytokines and increased glial activation within these brain structures (Chu et al., 2012; Dellarole et al., 2014; Marcello et al., 2013; Mor et al., 2010; Narita et al., 2006; Norman et al., 2010b; Ren et al., 2011; Takeda et al., 2009; Taylor et al., 2015; Wang et al., 2013a; Wu et al., 2014) (see Table 1). In addition there is emerging evidence that neuroinflammation in these forebrain and midbrain regions leads to the expression of affective disturbances (Burke et al., 2013a; Dellarole et al., 2014; Mor et al., 2010; Narita et al., 2006; Nascimento et al., 2015; Norman et al., 2010b; Ren et al., 2011; Taylor et al., 2015; Wang et al., 2013a; Wu et al., 2014) (see Table 2). It should be noted that in the mPFC and the hippocampus, as well as in the ventral posterolateral thalamus and brainstem structures (i.e., the locus coeruleus, rostroventral medulla, dorsal column nuclei and red nucleus), nerve injury-induced neuroinflammation has been causally linked with allodynia and hyperalgesia (Chiang

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et al., 2013; Covey et al., 2000; del Rey et al., 2011; Gosselin et al., 2010; Guo et al., 2012; Huang et al., 2014; Ignatowski et al., 1999; Ignatowski et al., 2005; LeBlanc et al., 2011; Li et al., 2008; Liu et al., 1995; Spengler et al., 2007; Tsai et al., 2012; Wang et al., 2015a; Wang et al., 2008; Wang et al., 2012; Wei et al., 2008; Zeng et al., 2014). With that said, the role of supraspinal neuroinflammation in the development of sensory dysfunction after nerve injury is not the focus of this review, and thus will not be discussed in detail. We hypothesise that peripheral nerve injury produces a specific pattern of supraspinal neuroinflammation that provides a basis for modulation of the neuroanatomical circuits controlling affective behaviours, thus contributing to the affective disturbances associated with neuropathic pain states. We will now examine the hippocampus, mPFC, striatum/NAcc, VTA, hypothalamus, amygdala and PAG in turn, briefly considering the role of each in affective disturbances after nerve injury before discussing how neuroinflammation can influence these affective disturbances.

4.1 Hippocampus The hippocampus modulates cognition and emotional state with dorsal and ventral functional networks influencing these two aspects of behaviour respectively (for detailed review see Fanselow and Dong, 2010; Moser and Moser, 1998). Hippocampal neurons are activated by noxious stimuli and their role in pain perception and awareness is longstanding (Dutar et al., 1985; Khanna and Sinclair, 1989). Recent preclinical studies have identified structural and functional changes in the hippocampus following experimental nerve injury (Cardoso-Cruz et al., 2011; Dellarole et al., 2014; Dimitrov et al., 2014; Hu et al., 2010; Kalman and Keay, 2014; Kodama et al., 2007; Mutso et al., 2012; Tanabe et al., 2008; Taylor et al., 2014;

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Terada et al., 2008). These nerve injury evoked changes in hippocampal anatomy and function appear to underpin disturbances in hippocampal-dependent cognition (Cardoso-Cruz et al., 2013; Dimitrov et al., 2014; Gregoire et al., 2012; Hu et al., 2010; Kodama et al., 2011; Leite-Almeida et al., 2012; Mutso et al., 2012; Ren et al., 2011; Wang et al., 2013a) and emotion (Dimitrov et al., 2014; Fukuhara et al., 2012; Gregoire et al., 2012; Hu et al., 2010; Leite-Almeida et al., 2012; Mutso et al., 2012; Nascimento et al., 2015; Wang et al., 2015b).

Over the last 15 years it has emerged that there is a robust upregulation of proinflammatory cytokines, TNF, IL-1β and IL-6 in the hippocampi of neuropathic rodents, strongest at 1-2 weeks and persisting beyond 3 weeks after injury (Al-Amin et al., 2011; Covey et al., 2000; del Rey et al., 2011; Gerard et al., 2015; Ignatowski et al., 1999; Ren et al., 2011; Sud et al., 2008; Uceyler et al., 2008). The cellular origins of hippocampal cytokines have yet to be fully evaluated, however nerve injury is known to increase the expression of TNF in hippocampal neurons (Spengler et al., 2007; Wang et al., 2013a). A glial origin is also possible although evidence is limited to only a single study showing SNL increased the expression of the astrocyte activation marker GFAP but only when combined with early life stress in male rats, whilst common peroneal nerve ligation did not alter microglia density or phenotype (Burke et al., 2013b; Zhang et al., 2008).

Regardless of the cellular origin, there is growing evidence that TNF, IL-1β and IL-6 are involved in altered hippocampal function and consequent affective disturbances after nerve injury. TNF in particular has emerged as a key player in injury-evoked impairment of LTP, reduced neurogenesis and decreased plasticity, evidenced through

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reduced expression of synaptic and myelin proteins (Dellarole et al., 2014; Gerard et al., 2015; Ren et al., 2011; Spengler et al., 2007; Sud et al., 2008; Wang et al., 2013a). Maternal deprivation (MD) combined with nerve injury exacerbated allodynia and increased expression of IL-6 and TNF in females rats, whilst in male rats there was an increase in GFAP and IL-1β as well as an increase in anxiety-like behaviour (Burke et al., 2013b). Similarly, early-life stress has been demonstrated as a risk factor for chronic pain clinically (Davis et al., 2005).

Hippocampal TNF and IL-1β are known to modulate glutamatergic signalling adversely affecting cognition. TNF alone enhances the expression of AMPA receptors lacking GluR2 expression leading to enhanced calcium currents, whilst TNF reduces the currents induced by glutamate, NMDA, AMPA and kainate (Beattie et al., 2002; Furukawa and Mattson, 1998; Stellwagen et al., 2005; Wang et al., 2013a). IL-1β reduces spontaneous excitatory post-synaptic currents in hippocampal neurons, but enhances NMDA-mediated currents that promote increased neurotoxicity (Viviani et al., 2003; Yang et al., 2005). Hippocampal LTP via NMDA-receptors underpins new memory formation, however TNF and IL-1 have well-established inhibitory effects on LTP (Butler et al., 2004; Cunningham et al., 1996; Tancredi et al., 1992). Thus is it perhaps unsurprising that elevated TNF in the dorsal hippocampus of nerve-injured rats suppresses NMDA mediated-currents, reducing LTP and consequently leading to impaired short-term memory (Ren et al., 2011; Wang et al., 2013a).

Neuroinflammation also appears to modulate affective behaviours via NMDA receptors. TNF-induced development of depressive-like behaviour is reversed by agmatine, an endogenous amine that blocks NMDA receptors (Neis et al., 2014). In

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neuropathic rats NMDA receptor activation with D-serine was anxiolytic, which may be explained by reduced expression of NR1 and NR2B subunits in the ventral hippocampus in the neuropathic setting (Wang et al., 2015b). Interestingly, D-serine had no effect on pain sensitivity, which is supportive of the idea that NMDAblockade not activation reduces pain (Sarkis et al., 2011; Wang et al., 2015b). NMDA receptor function is clearly implicated in mediating hippocampal-dependent cognitive function and affective behaviours, and nerve injury evoked TNF is a strong candidate to mediate NMDA receptor dysfunction.

Hippocampal neurogenesis is integral for affective state regulation (Sahay and Hen, 2007), however hippocampal TNF has been shown to inhibit this process (Cacci et al., 2005; Monje et al., 2003). In the context of nerve injury increased hippocampal TNF, acting on TNFR1, results in reduced neurogenesis and plasticity, as well as anhedonia and depressive-like behaviours (Dellarole et al., 2014; Nascimento et al., 2015). These behaviours were normalised by fluoxetine and thalidomide treatments that reduced TNF. Pro-inflammatory cytokines may contribute to a reduction in neurogenesis by suppression of the critical neurotrophic factor BDNF. Low levels of BDNF in the hippocampus are associated with stress, depression and pain (Duric and McCarson, 2005; Duric and McCarson, 2006; Duric and McCarson, 2007; Fukuhara et al., 2012). IL-1β reduces the production of BDNF, impairs neurogenesis and leads to sedentary behaviour and anhedonia (Goshen et al., 2008; Koo and Duman, 2008). In neuropathic rats elevated IL-6 expression mirrored a decrease in BDNF, both of which were reversed by blocking NMDA receptors (Al-Amin et al., 2011). Further neuropathy-induced impaired spatial memory, depressive-like behaviour and decreased BDNF expression were all normalised by amitriptyline, which is known to

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reverse elevated hippocampal TNF (Hu et al., 2010; Sud et al., 2008). It should be noted however, elevated spinal BDNF is pronociceptive after nerve injury (Coull et al., 2005), thus BDNF may not be a suitable target to normalise neuropathic symptoms, owing to different functions across the neuraxis.

NF-κB is a transcription factor activated by TNF and other cytokines. It is heavily involved in the transcription of genes encoding pro-inflammatory cytokines, and after nerve injury its role in pain facilitation at the spinal cord level is well documented (Ledeboer et al., 2005). Nerve injury induces an increase in hippocampal expression of the NF-κB subunit p65 that is ameliorated by NMDA receptor blockade (Chou et al., 2011). Activation of NF-κB also reduces BDNF resulting in impaired neurogenesis and depressive-like behaviours (Goshen et al., 2008; Haydon and Carmignoto, 2006; Koo and Duman, 2008). Thus, NF-κB may be a critical molecule in modulating signalling through NMDA receptors and suppressing BDNF expression. In summary, neuroinflammation in the neuropathic hippocampus is likely to influence affective behaviours by modulation of NMDA receptor function and/or reduction of neurogenesis via suppression of BDNF, whilst inhibition of LTP, also via NMDA receptors, is the likely mechanism of impaired memory.

4.2 Prefrontal cortex The rat prefrontal cortex (PFC) is an area of association cortex that predominantly consists of the mPFC and the orbitofrontal cortex (OFC) and regulates cortical control of visceral functions, as well as taking part in high-level cognitive and emotional processing (Ongur and Price, 2000). In the PFC nerve injury has been shown to induce anatomical and epigenetic changes, as well as dysregulation of glutamatergic

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signalling (Metz et al., 2009; Seminowicz et al., 2009; Tajerian et al., 2013). Of the mPFC sub-divisions three have been linked with affective disturbances in neuropathic conditions, they are from dorsal to ventral, the anterior cingulate (ACC), the prelimbic (PL) and infralimbic (IL) cortices. These nerve injury evoked changes appear to underpin disturbances in ACC-dependent cognition and motivation (Cardinal et al., 2003; LaBuda and Fuchs, 2005; LaGraize et al., 2004; Qu et al., 2011; Rajasethupathy et al., 2015; Walton et al., 2003) as well as PL/IL-dependent cognition and emotion (Giordano et al., 2012; Roozendaal et al., 2004). Currently, there is a lack of evidence that the OFC is involved in affective disturbances after nerve injury.

In analysing the PFC changes in neuroinflammation after nerve injury most of the early studies used a large tissue block (Apkarian et al., 2006; Liu et al., 2007; Uceyler et al., 2008), whilst more recent studies have focused on sub-divisions of the mPFC or the OFC (Al-Amin et al., 2011; Fuccio et al., 2009; Giordano et al., 2012; Shao et al., 2015). The consensus of these studies has been that nerve injury induces a proinflammatory response in the PFC that is most apparent 1-2 weeks post-injury and could be responsible for the neuroplastic changes described above. Moreover, there is growing evidence that PFC neuroinflammation is causally related to affective disturbances. Nerve injury induced elevation of IL-1β in the PFC results in depressive-like behaviour, which could be reversed by i.c.v. injection of either IL-1ra or oxytocin, with the latter attenuating IL-1β expression (Norman et al., 2010a; Norman et al., 2010b). Elevated TNF in the PFC also leads to depressive-like symptoms, both of which can be reversed by thalidomide or fluoxetine treatment (Nascimento et al., 2015). Chronic minocycline treatment led to a reduction in pro-

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inflammatory M1 microglia in the PFC of nerve-injured rats, whilst promoting polarisation to the anti-inflammatory M2 microglia in nerve-injured and olfactory bulbectomised (OB) depressed-rats (Burke et al., 2014). Despite the two different effects on microglia in the presence or absence of a depressive phenotype, minocycline reduced sensory disturbances in both cases, as well as normalising depressive-like behaviour in OB-SNL rats (Burke et al., 2014).

The ACC has been the focus of many studies due to its importance in encoding the affective component of pain, and nerve injury has been shown to increase IL-1β and IL-6 expression and reduce BNDF expression in this region (Al-Amin et al., 2011). Although these changes have not been directly linked to disruption in affective behaviours they were prevented by NMDA receptor blockade. Given that activation of NMDA receptors in the ACC mediates conditioned-place aversion (CPA) and supresses BNDF synthesis (Johansen and Fields, 2004; Kim et al., 2009), it is likely expression of CPA, upregulation of inflammatory mediators and suppression of BDNF are all dependent on NMDA receptor activation in the ACC.

Glial activity in the ACC has been directly linked to glutamate signalling and modulation of affective behaviours, with ipsilateral ACC astrocyte activation accompanied by anxiety-like behaviour 4 weeks after PSNL (Narita et al., 2006). Further investigation revealed ACC changes in astrocyte morphology led to increased glutamate and decreased GABA, and that astrocyte activation was causally related to sleep disturbances post-injury (Narita et al., 2011; Yamashita et al., 2014). Moreover, nerve injury enhances escape/avoidance behaviour, which was significantly inhibited via activation of GABAA receptors in the ACC (LaGraize et al., 2004).

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In the PL/IL, nerve injury increased IL-1β expression in astrocytes and IL-1R1 expression in glutamatergic neurons, alongside elevated extracellular glutamate, increased expression of vesicular and glial glutamate and GABA transporters (de Novellis et al., 2011; Giordano et al., 2012; Marcello et al., 2013). Altered fearresponding as a consequence of nerve injury-induced changes in glutamate neurotransmission in the PL/IL could indeed be driven by pro-inflammatory cytokines, since TNF, IL-1β and IFNγ have previously been shown to increase glutamate release from astrocytes (Ida et al., 2008).

Clearly there is a complex bi-directional modulation between pro-inflammatory cytokines and several neurotransmitter systems in the mPFC, however elevated glutamate possibly leading to cell death, decreased GABA and BDNF, and cytokine induction downstream of NMDA receptors are some common features. Further, there is increasing evidence that neuroinflammation in the neuropathic PFC leads to affective disturbances, including anxiety- and depressive-like behaviours as well as disruption to sleep/wake cycles.

4.3 Striatum and Nucleus accumbens The dorsal striatum comprises the caudate and putamen in humans, a single structure in the rat, and is primarily involved in action selection of cognitive, motor and oculomotor functions (Alexander et al., 1986), whereas the ventral striatum largely consists of the NAcc, which regulates motivational drive and goal-directed behaviour (Brischoux et al., 2009; Salamone et al., 2007; Sugam et al., 2012).

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Nerve injury alters the functional connectivity of the NAcc (Baliki et al., 2014; Chang et al., 2014), as well as myriad changes within the dopaminergic system (Austin et al., 2010; Chang et al., 2014; Sagheddu et al., 2015; Taylor et al., 2015; Wu et al., 2014). Nerve injury also impairs motivation and emotion, which are associated with changes in the glutamatergic system of the NAcc (Goffer et al., 2013; Marcello et al., 2013; Schwartz et al., 2014).

Nerve injury has been reported to produce a pro-inflammatory cytokine response in both aspects of the striatum. TNF is increased bilaterally in the NAcc of rats a week after SNI (Wu et al., 2014). In the contralateral striatum/thalamus, IL-1β mRNA is decreased 10 days after CCI and SNI, however by day 24 there is an increase in the SNI group (Apkarian et al., 2006). Another study reported a bilateral increase in striatal IL-1β protein expression 2 weeks after both SNI and CCI, changes which are co-incident with a reduction in striatal BDNF (Al-Amin et al., 2011). Further blockade of accumbal NMDA receptors, or activation of dopamine receptors attenuated the increase in IL-1β, as well as normalising BDNF levels and allodynia (Al-Amin et al., 2011; Sarkis et al., 2011).

Although the NAcc is critical for regulating affective disturbances after nerve injury there is only one study that conclusively demonstrates the involvement of neuroinflammation. TNF was found to be upregulated in the NAcc after SNI, alongside increased DAT expression and decreased synaptic dopamine levels (Wu et al., 2014). Behaviourally this was associated with prevention of conditioned place preference (CPP) to low dose morphine, indicating a reduction in reward-seeking behaviour. Moreover, neutralising TNF or knocking out TNFR1 normalised both

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DAT expression and synaptic dopamine levels, and reinstated CPP. Thus, post-injury accumbal release of TNF acting via TNFR1 appears to play a role in reduced motivation (Wu et al., 2014). Elevated striatal or accumbal cytokines may play additional roles in affective behaviours, given their ability to modulate both glutamatergic and dopaminergic signalling, both of which are disrupted and associated with affective disturbances after nerve injury in these brain regions.

4.4 Ventral tegmental area (VTA) The VTA is a crucial part of the brains reward circuitry (Lammel et al., 2011; Lammel et al., 2014; Lammel et al., 2012). Nerve injury impairs the opioid and dopaminergic systems of the VTA, which results in disturbances in VTA-dependent motivation (Ewan and Martin, 2011a, b; Martin et al., 2007; Ozaki et al., 2002; Taylor et al., 2015; Wu et al., 2014).

Based on the single study to investigate expression of pro-inflammatory cytokines in the VTA there was no change in TNF expression a week after nerve injury (Wu et al., 2014). Peripheral nerve injury, where the sciatic nerve is encased in polyethylene tubing, has however been shown to increase microglial activation in the VTA, leading to decreased opioid- and cocaine-stimulated dopamine responses resulting in blockade of CPP (Taylor et al., 2015). The authors suggest this occurs through loss of expression of the chloride transporter, KCC2, in GABAergic interneurons of the VTA, which leads to disinhibition of their projection targets. This study therefore highlights the potential of microglial inhibitors such as minocycline to normalise affective disturbances in the neuropathic state. Further studies are required to

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elucidate the exact mechanism of microglial-neuronal interaction, as TNF does not appear to be involved (Wu et al., 2014).

4.5 Amygdala The amygdala comprises a critical part of the circuitry regulating negatively valenced emotion, and is also implicated in cognition and motivation (Amano et al., 2010; Cai et al., 2014; Fanselow and Poulos, 2005; Janak and Tye, 2015; Jennings et al., 2013; Kim et al., 2013; Knobloch et al., 2012; Orsini et al., 2015; Penzo et al., 2014; Petrovich et al., 2001; Stefanik and Kalivas, 2013). Nerve injury leads to anatomical changes within the amygdala (Goncalves et al., 2008), as well as changes in glutamatergic and GABAergic activity that is co-incident with alterations in motivation and emotion (Ansah et al., 2010; Pedersen et al., 2007).

Few studies have examined changes in neuroinflammation in the neuropathic amygdala and those that have yielded mixed results. Reactive astrogliosis, but no change in microglial activity, density or phenotype, has been reported a week after nerve injury (Marcello et al., 2013; Zhang et al., 2008). Astrogliosis does occur alongside increased glial glutamate transporter-1, glial glutamate aspartate reuptake transporter and vesicular glutamate transporter 1, at a time-point when anxiety-like behaviour is evident, however no causal relationship has been demonstrated (Burke et al., 2013a; Marcello et al., 2013). By three weeks after nerve injury there is a predominantly anti-inflammatory response, with an increase in IL-10 and a decrease in IL-6 gene expression, with no change in IL-1β, TNF, GFAP and IBA-1 (Burke et al., 2013a). Further studies are urgently needed to investigate whether amygdala astrogliosis and a delayed anti-inflammatory response are involved in affective

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disturbances, possibility through dysregulation of glutamatergic and GABAergic systems also reported in the neuropathic amygdala.

4.6 Hypothalamus The hypothalamus plays an essential role in coordinating neuroendocrine, autonomic, and behavioural responses, as well as the sleep/wake cycle (for detailed review see Swanson, 2000). The hypothalamus and HPA axis are important in regulating affective behaviours after nerve injury (Norman et al., 2010b). Nerve injury has been reported to increase corticosterone levels (Kilburn-Watt et al., 2010), although contrastingly no changes in hypothalamic neuronal activation markers or CRH expression in basal or stress stimulatory conditions were reported (Bomholt et al., 2005; Ulrich-Lai et al., 2006). Nerve injury has been shown to lead to disturbances in the hypothalamic-pituitary-thyroid (HPT) axis in a sub-group of rats with persistent disability in social interactions after CCI (Kilburn-Watt et al., 2010, 2014).

Relatively few studies have examined expression of pro-inflammatory cytokines in this region in neuropathic rodents, yielding largely no change or a reduction in cytokine expression (Kilburn-Watt et al., 2014; Uceyler et al., 2008). A role for hypothalamic neuroinflammation in modulating nerve injury induced affective disturbances is limited, given the expression of disability in social interactions and dysregulation of the HPT axis were unrelated to changes in hypothalamic IL-1β and TNF measured 7 days after CCI (Kilburn-Watt et al., 2014). Although previous studies using peripheral immune activation demonstrated increased hypothalamic IL1β expression was capable of supressing the HPT axis at much earlier time points (Boelen et al., 2004; Boelen et al., 2006; Kakucska et al., 1994). Moreover, an

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increase in hypothalamic microglial activation was reported 4 days after CCI, which was blocked by an NMDA receptor antagonist (Takeda et al., 2009). Thus it remains a possibility that expression of hypothalamic neuroinflammation in the first few days following nerve injury may lead to affective disturbances.

4.7 Periaqueductal gray The PAG mediates descending pain modulation and integrated emotional coping strategies, as well as sleep-wake cycles (Bandler and Shipley, 1994; Gerashchenko et al., 2003; Gerashchenko et al., 2001; Gerashchenko and Shiromani, 2004; Keay and Bandler, 2001; Lu et al., 2006). In the PAG nerve injury has been shown to alter glutamatergic signalling (Marcello et al., 2013; Terashima et al., 2012), an effect which appears to be specific to the ventrolateral PAG column (vlPAG) (Ho et al., 2013; Renno, 1998).

There are mixed findings regarding neuroinflammation in the PAG after nerve. Firstly, nerve injury in rats increases expression of TNF, IL-1β and IL-6 in the PAG, effects which were reversed by a selective inhibitor of melanocortin-4 receptor, which also increased IL-10 and normalised sensory disturbances, perhaps through altered descending pain modulation (Chu et al., 2012). Whereas in mice, there was no change in IL-1β, IL-6 or TNF expression after SNI (Norman et al., 2010a). PSNL and CCI lead to increased microglia expression 4 and 6 days after injury, whereas a week after SNI and common peroneal nerve ligation there was no change in microglia density or activation (Marcello et al., 2013; Takeda et al., 2009; Zhang et al., 2008). Increased expression of astrocyte activation markers has been demonstrated in the PAG a week after CCI, however there have been conflicting results following SNI (Chu et al.,

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2012; Marcello et al., 2013; Mor et al., 2010; Norman et al., 2010b). Glial activation may underlie some changes in affective behaviours after SNI, as increased GFAP in the PAG occurred in mice with depressive-like behaviour (Norman et al., 2010b). Studies by Mor and colleagues have demonstrated columnar specificity of neuroinflammation with increased GFAP as well as evidence of cell death localised to the lateral PAG and caudal vlPAG columns of the PAG in a sub-group of rats that show persistent disability in social interactions and sleep disturbances after CCI (Monassi et al., 2003; Mor et al., 2011; Mor et al., 2010). This same sub-group of rats have decreased glucocorticoid receptor (GR) within the vlPAG column that is responsible for passive coping strategies and sleep regulation, but increased GR in the dorsolateral PAG, which mediates active coping strategies in response to psychological stressors (Mor and Keay, 2013). Therefore anatomically specific glial and neuronal maladaptations in the PAG after nerve injury appear to drive affective and sleep disturbances in this subgroup of rats. Thus, the best evidence for the involvement of neuroinflammation in the PAG with affective disturbances are the glial activation patterns specific to the vlPAG.

5. Anti-inflammatory treatments in neuropathic pain Anti-inflammatory treatments which interfere with pro-inflammatory cytokines or block glial activation have shown success in reducing allodynia and hyperalgesia in preclinical neuropathic pain models, and blockers of TNF, IL-1β and IL-6 are all effective in treating inflammatory pain conditions, such as rheumatoid arthritis (for detailed review see Austin and Moalem-Taylor, 2010; Austin and Moalem-Taylor, 2013; Kwilasz et al., 2015). Clinically, numerous studies have reported that patients with painful neuropathy have a lower expression of anti-inflammatory cytokines

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within the CSF and blood (Backonja et al., 2008; Uceyler et al., 2007), which suggests that an insufficiency of anti-inflammatory cytokines may propagate ongoing inflammation as well as clinical symptoms associated with the neuropathic pain state. Therefore, increasing physiological levels of anti-inflammatory cytokines may be a successful strategy to reduce inflammation and the associated sensory and affective disturbances in the neuropathic pain state. Despite this several randomised controlled drug trials have reported a lack of efficacy of novel anti-inflammatories to treat neuropathic pain (Bramson et al., 2015; Kalliomaki et al., 2013; Knezevic et al., 2015; Landry et al., 2012). This poor translational outcome is likely due in part to the problematic nature of drug delivery, which usually has a transient efficacy and can display unwanted side effects associated with the higher doses needed to effectively block the pain response. Further, in preclinical models anti-inflammatories are administered in the acute phase shortly after nerve injury when the contribution of pro-inflammatory mediators to allodynia and hyperalgesia are greatest, whereas in patients with established chronic pain (>6 months) the contribution of peripheral inflammatory mediators is diminished. Recent developments in anti-inflammatory cytokine-based gene therapies provide a novel approach to increasing the physiological expression of anti-inflammatory cytokines for a prolonged period of time, and have been found to be highly efficacious in preclinical models of chronic pain (for detailed reviews on anti-inflammatory gene therapies in chronic pain see Goins et al., 2012; Kwilasz et al., 2015). Of particular note, encapsulating plasmid DNA encoding IL-10 in biodegradable polymer-based micro-particles has been successful in producing sustained therapeutic effects, requiring a fraction of the dose of plasmid DNA encoding IL-10 compared to when the plasmid DNA was injected naked (Sloane et al., 2009; Soderquist et al., 2010). XT-101 and XT-150,

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encapsulated IL-10 gene therapy products developed by Xalud Therapeutics, appear to be the leading candidates for the delivery of IL-10 for prolonged periods and treatment for neuropathic pain, though results from clinical trials are not yet available.

Differences in the time-course and anatomical location of peripheral inflammation compared to supraspinal neuroinflammation in neuropathic pain states enhance the possibility that dampening the latter may successfully alleviate affective disturbances. The preclinical findings presented throughout this review confirm that assertion, however clinically the effectiveness of anti-inflammatory treatments to reduce affective disturbances associated with neuropathic pain have yet to be investigated. With that said randomised controlled trials have proven anti-inflammatories to be effective in treating depression. The cyclooxygenase-2 inhibitor, celecoxib, is effective in combination with antidepressants (Abbasi et al., 2012; Akhondzadeh et al., 2009; Majd et al., 2015; Muller et al., 2006), whilst the cytokine blocker, infliximab a soluble TNF antibody, is effective in treating major depressive disorder in a sub-group of patients with elevated C-reactive protein (Raison et al., 2013). Minocycline, which blocks microglial activation, has shown some early promise in the treatment of several neuropsychiatric disorders when combined with antidepressants or antipsychotics, although randomised controlled trials have yet to be completed (Dean et al., 2012; Miyaoka et al., 2012; Miyaoka et al., 2008).

Interestingly, there is preclinical evidence that dampening neuroinflammation in affective brain regions leads to a reduction in sensory disturbances, although affective disturbances were not tested in these studies (Covey et al., 2000; del Rey et al., 2011; Ignatowski et al., 1999; Ignatowski et al., 2005; Spengler et al., 2007). These findings

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indicate either a dual role for these regions in regulating both affective and sensory aspects of pain, or that reversal of affective disturbances improves the perception of sensory disturbances. Future studies whether clinical or preclinical should therefore examine both affective and sensory disturbances when exploring the role of neuroinflammation in neuropathic pain to delineate the specific mechanisms of each. Although further research is required the exciting possibility remains that aggressively targeting supraspinal inflammatory mediators in appropriate clinical populations, either alone, or in combination with current first-line treatments, may be efficacious in treating debilitating affective disturbances associated with neuropathic pain states.

6. Conclusions Following our systematic evaluation of the preclinical evidence, we assert that the emergence of affective disturbances in neuropathic pain states are contingent on altered inflammatory mediators in the interconnected hippocampal-medial prefrontal networks that subserve affective behaviours. Further our recent preclinical findings suggest that an individual’s immune reactivity could predispose them to pain and affective disturbances. Therefore, certain clinical populations may be more responsive to potential anti-inflammatory treatments.

After nerve injury transmission of peripheral inflammation to the brain through a set of well-defined immune-to-brain signalling pathways likely leads to supraspinal neuroinflammation. Nerve injury evokes an anatomically specific pattern of neuroinflammation. We believe neuroinflammation in the interconnected hippocampal-mPFC circuitry is critical in the development of depression, anhedonia,

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cognitive impairment and sleep disturbances that are a common feature of neuropathic pain in both preclinical and clinical settings. It is also possible that once neuroinflammation is established in these heavily interconnected circuits it may be capable of self-amplification by volume diffusion or neural transmission that may underlie the entrenchment of affective disturbances.

There are a variety of compelling mechanisms by which nerve injury induced neuroinflammation modulates neural circuits controlling affective behaviours. The strongest evidence is for modulation of the glutamatergic system, either directly via elevated glutamate levels, altered expression of NMDA and AMPA receptors and glutamate transporters, or indirectly through the metabolites of the IDO pathway. Major consequences of this are disruption of LTP, which has been linked to cognitive deficits, whilst metabolites of the IDO pathway and elevated glutamate have been linked with neurotoxicity and depression. Suppression of critical brain neurotrophic factors, such as BDNF, by cytokines may contribute to a reduction in neurogenesis and neuroplasticity, processes which are diminished in depression. TNF modulation of dopaminergic systems has been associated with blunted reward, whilst midbrain glial activation appears to contribute to midbrain cell death and altered expression of glucocorticoids that is associated with affective disturbances.

Despite mounting evidence of a causal relationship between neuroinflammation in affective brain regions after peripheral nerve injury and affective disturbances preclinically, a greater appreciation of several key areas is still required. In order to fully support the notion that nerve injury-evoked inflammatory mediators in brain regions associated with affective behaviours underpin affective disturbances, a more

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detailed anatomical examination of their expression patterns and cellular origin is necessary. Further, the exact timeline for the appearance of supraspinal neuroinflammation in different regions remains to be elucidated. To address this, longitudinal studies examining the magnitude of neuroinflammation throughout the brain in several nerve injury models combined with thorough assessment of sensory and affective disturbances are required. These preclinical studies should be combined with longitudinal brain imaging studies in patients with neuropathic pain that take advantage of new imaging techniques that can examine glial activation patterns(Alshelh et al., 2016; Loggia et al., 2015). Developing a greater appreciation of the precise molecular mechanisms and anatomical circuits through which neuroinflammation regulates specific facets of affective behaviour in neuropathic pain states should also be a priority. Understanding these key issues is critical to guide the development of novel strategies to target specific supraspinal inflammatory mediators at critical time-points in appropriate clinical populations, thus enhancing the potential for efficacious treatments of the usually treatment-resistant affective disturbances.

Acknowledgements The authors would like to thank Dr. F.R. Walker for his critical appraisal and insight on an earlier version of the manuscript.

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Figure legends Figure 1. A schematic overview showing three proposed routes of immune-to-brain signalling which can lead to brain neuroinflammation following a peripheral immune challenge. (a) Neural transmission; the most common example is via the vagus nerve, where IL-1β activates vagal afferents, altering the firing pattern of neurons in the nucleus tractus solitarius (NTS) and its projection targets the amygdala and hypothalamus, leading to the induction of cytokine release by neighbouring glial cells (Brady et al., 1994; Goehler et al., 1999; Hansen et al., 2001). (b) Humoral transmission; pro-inflammatory cytokines enter systemic circulation and are either actively transported across the BBB, pass through into CVOs or act on astrocytes and endothelial cells within the BBB to induce their production in the brain, either directly or indirectly via the release of nitric oxide (NO) and PGs (Banks et al., 1994; Banks et al., 1991; Konsman et al., 1999; Konsman et al., 2002; Pan and Kastin, 2002; Plotkin et al., 1996; Van Dam et al., 1996). (c) Molecular transmission; systemic cytokines, such as TNF, trigger the release of MCP-1 from microglia within the brain that facilitates monocyte adhesion and rolling along the endothelial cells, thus allowing monocyte infiltration across the BBB which results in the propagation of neuroinflammation (D'Mello et al., 2009; D'Mello et al., 2013; Mitchell et al., 2009).

Figure 2. A schematic overview of five possible mechanisms by which either peripheral inflammation or supraspinal neuroinflammatory mediators may underlie changes in affective behaviour. (i) Altered glutamatergic and GABAergic activity, resulting in a reduction in LTP and excitotoxicity resulting in neuronal and glial damage (Beattie et al., 2002; Bezzi et al., 2001; Furukawa and Mattson, 1998; Ida et al., 2008; Ren et al., 2011; Stellwagen et al., 2005; Tilleux et al., 2007; Wang et al.,

50

2013b; Yang et al., 2005). (ii) An increase in production of the enzyme, indoleamine 2,3-dioxygenase (IDO) that facilitates the conversion of tryptophan to kynurenine (KYN). In microglia KYN can be converted to the neurotoxic metabolite QUIN, an agonist of the NMDA receptor (Guillemin et al., 2001). Increased expression of KYN and QUIN has been linked to depression in humans, as well as depressive-like behaviour in neuropathic and inflammatory chronic pain models (Kim et al., 2012; O'Connor et al., 2009; Walker et al., 2013; Zhou et al., 2015). (iii) Suppression of growth factors, particularly BDNF, leading to a reduction in neurogenesis (Goshen et al., 2008; Goshen et al., 2007). (iv) Changes in monoamine turnover (Covey et al., 2000; Moron et al., 2003; Pellegrino et al., 2011; Wu et al., 2007; Zhu et al., 2010). (v) Reduced glucocorticoid release as well as a decrease in GR expression and function (Besedovsky and del Rey, 1996; Pace et al., 2007).

51

52

53

Table 1. Summary of previous studies on the time course of neuroinflammation following peripheral nerve injury. Brain region Time Nerve Species/Strain/ Neuroinflammation period injury Sex model Hippocampus <24 SNI Rat Increased TNF protein hours SpragueDawley Male CCI Mice Decreased TNF, IL-1β, and IL-4 mRNA C57BL/6J Female 3-4 days SNI Rat Increased TNF protein SpragueDawley Male 1-2 CCI and Rat Increased bilateral IL-1β, and increased weeks SNI Spraguecontralateral IL-6 protein after both CCI and Dawley SNI Female CCI Rat Increased TNF protein, which disappears at day Sprague14 Dawley Male CCI and Rat Increased contralateral IL-1β and IL-6 mRNA SNI Spragueafter SNI but not CCI in Wistar-Kyoto Dawley/ Increased contralateral IL-1β after both SNI and Wistar-Kyoto CCI but no change in IL-6 mRNA in SpragueMale Dawley CCI Rat Increased contralateral TNF protein SpragueDawley Male CCI Rat Increased TNF protein SpragueDawley Male Sciatic Mouse Increased TNF protein nerve Swiss crush Male SNI Rat Increased TNF protein SpragueDawley Male CCI Rat Increased TNF mRNA SpragueDawley Male CCI Rat Increased contralateral TNF protein SpragueDawley Male SNI Rat Increased TNF protein SpragueDawley Male Common Mouse No change in microglia activation peroneal Cx3cr1GFP/+

54

Reference

(Ren et al., 2011)

(Uceyler et al., 2008) (Ren et al., 2011)

(Al-Amin et al., 2011)

(Covey et al., 2000)

(del Rey et al., 2011)

(Gerard et al., 2015)

(Ignatowski et al., 1999)

(Nascimento et al., 2015) (Ren et al., 2011)

(Spengler et al., 2007)

(Sud et al., 2008)

(Wang et al., 2013)

(Zhang et al., 2008)

3+ weeks

nerve ligation SNL

CCI and SNI

SNI

SNI

PFC

<24 hours

CCI

1-2 weeks

CCI and SNI

CCI and SNI SNI

SNI

SNL

SNI

PSNL

Sciatic nerve crush SNI

SNI

SNI

Not stated Rat SpragueDawley Male Rat SpragueDawley/ Wistar Kyoto Male

Rat SpragueDawley Male Rat SpragueDawley Male Mice C57BL/6J Female Rat SpragueDawley Female Rat Wistar Kyoto Male Mouse C57BL/6N Male Mouse C57BL/6N Male Rat SpragueDawley Male Rat SpragueDawley Male Mouse C57BL/6J Male Mouse Swiss Male Mouse C57BL/6 Male Mouse C57BL/6 Male Rat Sprague-

Increased astrocyte expression when combined with MD

(Burke et al., 2013b)

Increased contralateral IL-1β but disappearance of increased IL-6 mRNA after SNI in Wistar Kyoto Increased contralateral IL-1β after SNI but disappearance of increased IL-1β mRNA after CCI, and increased IL-1ra mRNA after both SNI and CCI in Sprague-Dawley Increased TNF protein

(del Rey et al., 2011)

(Ren et al., 2011)

Increased TNF protein

(Wang et al., 2013)

Decreased TNF, but not IL-1β, mRNA

(Uceyler et al., 2008)

Increased bilateral IL-1β and contralateral IL-6 protein after both SNI and CCI (ACC)

(Al-Amin et al., 2011)

Increased contralateral IL-1β mRNA after SNI but not CCI

(Apkarian et al., 2006)

Increased IL-1β protein (OFC)

(Fuccio et al., 2009)

Increased IL-1R1 but not IL-1β protein (PL-IL)

(Giordano et al., 2012)

Increased contralateral IL-1β, IL-6 and TNF but not IL-10 protein

(Liu et al., 2007)

No change in astrocyte or microglia expression (mPFC)

(Marcello et al., 2013)

Increased astrocyte expression and activation, but no change in mRNA (ACC)

(Narita et al., 2011)

Increased TNF protein

(Nascimento et al., 2015)

Increased IL-1β but not IL-6, TNF and astrocyte mRNA when also socially isolated

(Norman et al., 2010a)

Increased IL-1β but not IL-6, TNF and astrocyte mRNA

(Norman et al., 2010b)

Increased contralateral IL-1β and IL-10 protein (VLO)

(Shao et al., 2015)

55

PSNL

3+ weeks

Common peroneal nerve ligation CCI and SNI PSNL

Striatum/ NAcc

1-2 weeks

CCI and SNI

CCI and SNI SNI

SNI

VTA

3+ weeks

CCI and SNI

1-2 weeks

PNI

SNI

Amygdala

1-2 weeks

3+ weeks

Hypothalamus

SNI

Common peroneal nerve ligation SNL

<24 hours

CCI

3-4 days

PSNL

1-2

CCI

Dawley Male Mouse C57BL/6J Male Mouse Cx3cr1GFP/+ Not stated

Increased astrocyte expression and activation after a thermal noxious stimulus (ACC)

(Yamashita et al., 2014)

No change in microglia activation (ACC and PFC)

(Zhang et al., 2008)

Rat Wistar Kyoto Male Mouse C57BL/6J Male Rat SpragueDawley Female Rat Wistar Kyoto Male Rat SpragueDawley Male Rat SpragueDawley Male Rat Wistar Kyoto Male Mouse C57BL/6J Male Rat SpragueDawley Male Rat SpragueDawley Male Mouse Cx3cr1GFP/+ Not stated

Disappearance of increased contralateral IL-1β mRNA after SNI, no change after CCI

(Apkarian et al., 2006)

Increased astrocyte expression (ACC)

(Narita et al., 2006)

Increased bilateral IL-1β after both CCI and SNI, and decreased contralateral IL-6 protein after CCI (Striatum)

(Al-Amin et al., 2011)

Decreased contralateral IL-1β mRNA after both CCI and SNI (Striatum/thalamus)

(Apkarian et al., 2006)

No change in astrocyte or microglia expression (NAcc)

(Marcello et al., 2013)

Increased bilateral TNF protein (NAcc)

(Wu et al., 2014)

Increased contralateral IL-1β after SNI and disappearance of decreased contralateral IL-1β mRNA after CCI (Striatum/thalamus) Increased microglial activation

(Apkarian et al., 2006)

Rat SpragueDawley Male Mice C57BL/6J Female Rat Wistar Male Rat

(Taylor et al., 2015)

No change in bilateral TNF protein

(Wu et al., 2014)

Increased astrocyte expression, but no change in microglia expression

(Marcello et al., 2013)

No change in microglia activation

(Zhang et al., 2008)

Increased IL-10 and decreased IL-6, but no change in IL-1β, TNF, astrocyte or microglia mRNA

(Burke et al., 2013a)

Decreased TNF and IL-4, but no change IL-1β, mRNA

(Uceyler et al., 2008)

Increased microglial expression

(Takeda et al., 2009)

No change in IL-1β or TNF mRNA

(Kilburn-

56

weeks

PAG

3-4 days

CCI

PSNL

1-2 weeks

CCI

SNI

CCI

SNI

Common peroneal nerve ligation

SpragueDawley Male Rat Wistar Male Rat Wistar Male Rat Wistar Male Rat SpragueDawley Male Rat SpragueDawley Male Mouse C57BL/6 Male Mouse Cx3cr1GFP/+ Not stated

Watt et al., 2014) Increased astrocyte, microglia, TNF, IL-1β and IL-6, but no change in IL-10 protein

(Chu et al., 2012)

Increased microglial expression

(Takeda et al., 2009)

Increased astrocyte, microglia, TNF, IL-1β and IL-6, but no change in IL-10 protein

(Chu et al., 2012)

No change in astrocyte or microglia expression

(Marcello et al., 2013)

Increased astrocyte expression (lPAG and vlPAG)

(Mor et al., 2010)

Increased astrocyte, but no change in IL-1β, IL6 and TNF mRNA

(Norman et al., 2010b)

No change in microglia activation

(Zhang et al., 2008)

57

Table 2. Summary of previous studies on the effects of neuroinflammation on affective behaviour post-injury. Brain Region Nerve Species/ Affective Key findings Reference injury Strain/ behavioural model Sex test Hippocampus SNI Mouse Sucrose Decreased sucrose preference (Dellarole et C57BL/6 preference causally related to an increase al., 2014) Not stated in TNF and TNFR1 signalling Hippocampus

SNI

Rat SpragueDawley Male Rat SpragueDawley Male Mouse Swiss Male

Radial maze Novel object recognition Novel object recognition

Impaired short-term spatial and novelty recognition memory causally related to an increase in TNF Impaired short-term novelty recognition memory causally related to an increase in TNF

Hippocampus

SNI

Hippocampus and PFC

Crush injury

mPFC (ACC)

FST TST

Increased immobility causally related to an increase in TNF

(Nascimento et al., 2015)

PSNL

Mouse C57BL/6J Male

Light-dark box EPM

(Narita et al., 2006)

mPFC (ACC)

PSNL

Mouse C57BL/6J Male

EEG/EMG

PFC

SNI

Mouse C57BL/6 Male

FST

Decreased time spent in light compartment and in open arms causally related to an increase in astrocytes Increased wakefulness and decreased NREM related to an increase in astrocyte expression and activation Increased immobility related to an increase in IL-1β and IL-1R1 signalling

NAcc

SNI

CPP

PNI

Prevention of CPP to low dose morphine causally related to an increase in TNF and TNFR1 signalling Prevention of CPP to low dose synthetic opioid causally related to an increase in microglial activation

(Wu et al., 2014)

VTA

Amygdala

SNL

Rat SpragueDawley Male Rat/Mouse SpragueDawley/ C57BL/6J Male/Male Rat SpragueDawley Male

(Burke et al., 2013a)

PAG

CCI

Rat SpragueDawley Male

Residentintruder paradigm

Decreased time spent in the inner zone related to an increase in IL-10 and decrease in IL-6 gene expression at a later time point Decreased dominance behaviour related to an increase in astrocyte expression within the vlPAG in a sub-group of rats

CPP

Open field

58

(Ren et al., 2011)

(Wang et al., 2013)

(Narita et al., 2011)

(Norman et al., 2010b)

(Taylor et al., 2015)

(Mor et al., 2010)

Highlights • Peripheral inflammation results in affective disturbances in neuropathic pain • Nerve injury induces increases in brain cytokine expression and glial cell activity • Inflammation in hippocampal-prefrontal circuits causes affective disturbances • Anti-inflammatories may prevent affective disturbances in neuropathic pain

59