Chronic at-level thermal hyperalgesia following rat cervical contusion spinal cord injury is accompanied by neuronal and astrocyte activation and loss of the astrocyte glutamate transporter, GLT1, in superficial dorsal horn

Highlights

• We characterized 2 rat models of unilateral cervical contusion spinal cord injury.

• Both models resulted in chronic thermal hyperalgesia in the ipsilateral forepaw.

• Spinothalamic pain transmission neurons were chronically activated in both models.

• Injury resulted in persistent astrocyte activation and reduced GLT1 expression.

• Histological changes were pronounced in superficial laminae of the dorsal horn.

Abstract

Neuropathic pain is a form of pathological nociception that occurs in a significant portion of traumatic spinal cord injury (SCI) patients, resulting in debilitating and often long-term physical and psychological burdens. While many peripheral and central mechanisms have been implicated in neuropathic pain, central sensitization of dorsal horn spinothalamic tract (STT) neurons is a major underlying substrate. Furthermore, dysregulation of extracellular glutamate homeostasis and chronic astrocyte activation play important underlying roles in persistent hyperexcitability of these superficial dorsal horn neurons. To date, central sensitization and astrocyte changes have not been characterized in cervical SCI-induced neuropathic pain models, despite the fact that a major portion of SCI patients suffer contusion trauma to cervical spinal cord. In this study, we have characterized 2 rat models of unilateral cervical contusion SCI that behaviorally result in chronic persistence of thermal hyperalgesia in the ipsilateral forepaw. In addition, we find that STT neurons are chronically activated in both models when compared to laminectomy-only uninjured rats. Finally, persistent astrocyte activation and significantly reduced expression of the major CNS glutamate transporter, GLT1, in superficial dorsal horn astrocytes are associated with both excitability changes in STT neurons and the neuropathic pain behavioral phenotype. In conclusion, we have characterized clinically-relevant rodent models of cervical contusion-induced neuropathic pain that result in chronic activation of both STT neurons and astrocytes, as well as compromise in astrocyte glutamate transporter expression. These models can be used as important tools to further study mechanisms underlying neuropathic pain post-SCI and to test potential therapeutic interventions.

Introduction

A significant portion of traumatic spinal cord injury (SCI) patients experience one or more forms of neuropathic pain, resulting in debilitating and often long-term physical and psychological burdens. These include enhanced responsiveness to noxious peripheral stimuli (hyperalgesia), painful sensation in response to formally innocuous peripheral stimuli (allodynia), and often spontaneous pain in the absence of peripheral stimulation (Hulsebosch et al., 2009).

Hyperexcitability of dorsal horn pain projection neurons (“central sensitization”) is a major substrate for neuropathic pain following SCI (Gwak and Hulsebosch, 2011b). This includes decreased threshold for activation (i.e. action potential generation), increased spontaneous activity, expansion of peripheral receptive field, and the occurrence of after-discharges in dorsal horn spinothalamic tract (STT) pain projection neurons (Latremoliere and Woolf, 2009). In particular, alterations in the properties and proportion of wide-dynamic range pain projection neurons (in both superficial and deeper laminae) occur following SCI. Furthermore, many of these changes in dorsal horn neuron properties are persistent, likely accounting for the chronic nature of neuropathic pain after SCI.

Dysregulation of extracellular glutamate homeostasis is thought to play a major mechanistic role in dorsal horn neuron hyperexcitability following SCI (Tao et al., 2005). Glutamate is the primary neurotransmitter released from primary afferent terminals onto 2nd order dorsal horn pain projection neurons. Following injury, elevated levels of extracellular glutamate can result from increased synaptic release by primary afferent pain fibers, release from injured neurons, axons and glial cells, and compromised glutamate clearance due to glutamate transporter dysfunction. Increased activation of glutamate receptors (particularly NMDA receptors) is mediated by elevated dorsal horn levels of extracellular glutamate detailed above, as well as by changes in the expression, localization and function (via phosphorylation, for example) of glutamate receptor subunits in these neurons (Bleakman et al., 2006). Additional mechanisms that might account for glutamate-mediated central changes in nociceptive neurotransmission in the dorsal horn after SCI include: (1) increased activation of glial cells such as astrocytes and microglia by glutamate (Cao and Zhang, 2008, Hansson, 2006, Ren, 2010), (2) decreased inhibitory tone due to glutamate-mediated excitotoxic loss of GABAergic interneurons (Gwak and Hulsebosch, 2011a), and (3) reduced recycling of glutamine from astrocytes back to inhibitory neurons for GABA synthesis because of decreased glutamate uptake and subsequent conversion of glutamate to glutamine in activated astrocytes (Ortinski et al., 2010).

In the CNS, glutamate is efficiently cleared from the synapse and from other sites by glutamate transporters located on the plasma membrane (Maragakis and Rothstein, 2004). Astrocytes are supportive glial cells that play a host of crucial roles in CNS function (Pekny and Nilsson, 2005). In particular, astrocytes express the major CNS glutamate transporter, GLT1, which is responsible for the vast majority of functional glutamate uptake in the CNS, particularly in the spinal cord (Maragakis and Rothstein, 2006). Studies have focused on therapeutically targeting glutamate receptor over-activation in SCI models. However, regulation of extracellular glutamate homeostasis by GLT1 following SCI has not been extensively addressed, particularly with respect to modulation of the excitability of dorsal horn neurons involved in pain neurotransmission. This is despite the fact that astrocyte GLT1 plays the central role in regulating extracellular glutamate homeostasis in the spinal cord (Maragakis and Rothstein, 2006).

Following SCI, astrocyte loss/dysfunction and/or altered GLT1 physiology can result in dysregulation of extracellular glutamate homeostasis (Lepore et al., 2011a, Lepore et al., 2011b), as well as further glial activation and consequent additional compromise in GLT1 function. Vera-Portocarrero et al. (2002) reported changes in GLT1 protein levels within 24 h following thoracic contusion, while Olsen et al. (2010) reported longer-term decreases in GLT1 expression after thoracic SCI. Our group has also demonstrated long-term decreases in GLT1 expression and functional GLT1-mediated glutamate uptake following thoracic contusion (Lepore et al., 2011a, Lepore et al., 2011b). As glutamate homeostasis dysregulation can persist after SCI, it is important to characterize and therapeutically target both early and longer-term changes in transporter expression and function, as well as to do so in the context of dorsal horn neuron hyperexcitability and neuropathic pain following cervical SCI.

Chronic glial activation contributes to the development and persistence of neuropathic pain following SCI. The anatomical mechanisms of hyperalgesia, allodynia and spontaneous pain differ; nevertheless, significant astrocyte activation occurs in all of these pain states, regardless of whether there is central SCI or peripheral nerve damage (Hansson, 2006). Activated astrocytes (and microglia) play key roles in neuropathic pain (Scholz and Woolf, 2007). Following SCI, elevation in the levels of factors such as glutamate, CGRP and substance P can activate astrocytes, resulting in astrocytic release of ATP, NO and pro-inflammatory cytokines that facilitate nociceptive neurotransmission by increasing excitability of dorsal horn pain neurons. These signaling molecules can also activate neighboring astrocytes to maintain and possibly anatomically spread pain to uninjured areas (Hulsebosch, 2008). Astrocyte activation can also compromise key physiological functions such as glutamate uptake (Lepore et al., 2011a), resulting in further dysregulation of extracellular glutamate homeostasis. As described above, dysregulation of glutamate signaling can itself promote dorsal horn neuron hyperexcitability and can also lead to additional astrocyte stimulation, both locally and in more distant locations. Furthermore, this overall gliopathic response can persist, possibly maintaining a chronic neuropathic state (Cao and Zhang, 2008).

Glutamate has been shown to play a central role in this astrocyte response and in neuropathic pain in general (Hulsebosch, 2008, Hulsebosch et al., 2009). Glutamate uptake inhibition or administration of glutamate receptor agonists to intact spinal cord results in spontaneous pain behaviors similar to those seen after SCI (Liaw et al., 2005). There is high level expression of AMPARs, NMDARs, mGluRs (and GLT1) in superficial dorsal horn (Bleakman et al., 2006), and a role for these glutamatergic receptor types has been documented in neuropathic pain using pharmacological agents targeting both ionotropic and metabotropic receptors (Bleakman et al., 2006, Mills et al., 2000, Mills et al., 2001, Mills et al., 2002a, Mills et al., 2002b, Mills and Hulsebosch, 2002). In models of chronic neuropathic pain induced by peripheral nerve injury, there are reduced dorsal horn GLT1 levels, and maintenance of GLT1 expression via drugs such as ceftriaxone and propentofylline can block neuronal hyperexcitability and the development of pathological pain (Hobo et al., 2011, Hu et al., 2010, Inquimbert et al., 2012, Nie and Weng, 2010, Tawfik et al., 2008). It is therefore crucial to address the underlying role of glutamate – and specifically the crucial role played by astrocytes – in breaking this “loop” of glial activation-neuronal hyperexcitability-neuropathic pain following SCI.

Importantly, studies on neuropathic pain need to model clinically-relevant aspects of human SCI. Injury to cervical spinal cord represents greater than half of all human SCI cases, in addition to often resulting in the most severe physical and psychological debilitation (Lane et al., 2008). Furthermore, contusion-type SCI predominates in humans (McDonald and Becker, 2003). Despite this make-up of the clinical population, the vast majority of animal studies have not employed cervical contusion models, and therapies targeting neuropathic pain have not been tested with cervical contusion. Although use of thoracic SCI animal models has predominated, cervical contusion SCI models have recently been developed (Aguilar and Steward, 2010; Gensel et al., 2006; Lee et al.; Sandrow-Feinberg et al. 2010; Sandrow-Feinberg et al., 2009; Sandrow et al., 2008; Stamegna et al., 2011), including our own (Nicaise et al., 2012a, Nicaise et al., 2012b, Nicaise et al., 2013).

In this study, we have characterized 2 clinically-relevant rat models of unilateral cervical contusion SCI. Given that astrocyte activation, loss of astrocyte GLT1 function and consequent dysregulation of extracellular glutamate homeostasis can contribute to the hyperexcitability of second order dorsal horn STT neurons following SCI, we have spatiotemporally examined (in the superficial dorsal horn) astrocyte and STT neuron activation, as well as alterations in GLT1 expression, in these models. We report that both SCI models result in the development and chronic persistence of at-level thermal hyperalgesia, as well as in chronic activation of STT neurons in the cervical spinal cord. Furthermore, persistent astrocyte activation and significantly reduced astrocyte GLT1 expression in superficial dorsal horn are associated both with changes in STT neurons and the neuropathic pain behavioral phenotype. Going forward, these models can be used as important tools to further study mechanisms underlying the development of neuropathic pain post-SCI and to test potential therapeutic interventions.