Elsevier

Experimental Neurology

Volume 212, Issue 2, August 2008, Pages 337-347
Experimental Neurology

Remote activation of microglia and pro-inflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats

https://doi.org/10.1016/j.expneurol.2008.04.009Get rights and content

Abstract

Spinal cord injury (SCI) impairs sensory systems causing chronic allodynia. Mechanisms underlying neuropathic pain have been more extensively studied following peripheral nerve injury (PNI) than after central trauma. Microglial activation, pro-inflammatory cytokine production and activation of p38 MAP kinase pathways may induce at-level allodynia following PNI. We investigated whether midthoracic SCI elicits similar behavioral and cellular responses below the level of injury (lumbar spinal cord; L5). Importantly, we show that anatomical connections between L5 and supraspinal centers remain intact after moderate SCI allowing direct comparison to a well-established model of peripheral nerve injury. We found that SCI elicits below-level allodynia of similar magnitude to at-level pain caused by a peripheral nerve injury. Moreover, the presence of robust microglial activation in L5 cord predicted allodynia in 86% of rats. Also increased phosphorylation of p38 MAP kinase occurred in the L5 dorsal horn of allodynic rats. For below-level allodynia after SCI, TNF-α and IL-1β increased in the L5 dorsal horn by 7 dpo and returned to baseline by 35 dpo. Interestingly, IL-6 remains at normal levels early after SCI and increases at chronic time points. Increased levels of pro-inflammatory cytokines also occurred in the thalamus after SCI-induced allodynia. These data suggest that remote microglial activation is pivotal in the development and maintenance of below-level allodynia after SCI. Fractalkine, a known activator of microglia, and astrocytes were not primary modulators of below-level pain. Although the mechanisms of remote microglial activation are unknown, this response may be a viable target for limiting or preventing neuropathic pain after SCI in humans.

Introduction

Over two-thirds of patients with spinal cord injury (SCI) have significantly diminished quality of life due to the presence and persistence of neuropathic pain (Widerstrom-Noga et al., 2001, Siddall et al., 2003). Hypersensitivity after SCI is categorized as above-, at- and below-level pain. Clinical and experimental classifications include: allodynia — the perception of an innocuous stimulus as painful; or hyperalgesia — exaggerated, heightened responses to a noxious stimulus (IASP, 2008). Previously, we and others showed that below-level pain develops after SCI in animal models (Kloos et al., 2005, Hutchinson et al., 2004, Lindsey et al., 2000, Christensen et al., 1996a, Siddall et al., 1995, Bruce et al., 2002, Yezierski and Park, 1993, Hains and Waxman, 2006).

Peripheral nerve injury models are useful for identifying mechanisms of neuropathic pain because aversive responses can be elicited without directly injuring CNS pain pathways. Activated glia (Coull et al., 2005, Tsuda et al., 2003), induction of select intracellular signaling pathways (e.g. p38; Jin et al., 2003) and robust increases in cytokine and chemokine synthesis (e.g. TNFα, fractalkine; Milligan et al., 2004, DeLeo et al., 1996, DeLeo et al., 2000, Sweitzer et al., 2001, Raghavendra et al., 2004) have all been associated with hind paw allodynia after peripheral nerve lesion. Although these cellular and molecular changes are common after SCI, whether they are associated with or are responsible for the onset and maintenance of SCI-induced neuropathic pain is unknown. In fact, few have studied mechanisms of SCI-induced neuropathic pain below the level of injury. Recent reports suggest that robust glial activation occurs at sites caudal to SCI in rats with below-level pain (Hains and Waxman, 2006, Nesic et al., 2005, Peng et al., 2006, Detloff et al., 2004). Additionally, increased expression of TNFα was recently described caudal to the level of SCI and was correlated with short-term allodynia (Peng et al., 2006). To further these observations and provide novel insight to mechanisms of below-level neuropathic pain induced by spinal contusion injury, we tested whether pain behaviors and accompanying glial activation were associated with the upregulation of p38 MAPK and/or fractalkine (CX3CL1). Importantly, these parameters were compared to a conventional model of peripheral nerve injury-induced neuropathic pain where the mechanisms of pain induction and maintenance have been better defined. By performing these lesions in parallel, we can begin to understand the cellular and molecular substrates responsible for SCI-induced pain development and maintenance. Finally, we determined whether pro-inflammatory cytokines are elevated in spinal cord segments removed from the primary site of SCI. Our data show that below-level pain elicited by moderate SCI was associated with a significant increase in microglial activation and pro-inflammatory cytokines 10 segments below the level of SCI. This work has been reported in abstract form (Detloff et al., 2004, Detloff et al., 2006, Detloff et al., 2007).

Section snippets

Subjects and surgeries

Sixty-two adult female Sprague–Dawley rats (205–231 g) were randomly assigned to six groups: Naïve (n = 12), laminectomy control (LAM; n = 4), Mild SCI (0.5 mm cord displacement; n = 5) or Moderate SCI (1.1 mm cord displacement; n = 29), L5 modified spinal nerve ligation (mSNL) with a short (14 day; mSNL14; n = 4) or long (35 day; mSNL35; n = 5) survival. The mSNL14 group was included to replicate the study by Tsuda et al. (2003) where activated microglia correlated with allodynic behavior. The mSNL35 rats

Mechanical allodynia

Although the mode and site of nerve trauma are markedly different between SCI and mSNL groups, by 21 dpo, all rats in the mSNL35 and the Moderate SCI groups exhibited allodynia of similar magnitude (Fig. 1). No rats demonstrated overt evidence of spasticity prior to or during sensory testing such as clonus, sustained anti-gravity posturing of the HL and resistance to velocity-dependent, passive joint movement. Importantly, allodynia persisted for the duration of the study in these groups

Discussion

The current work characterizes the cellular and molecular responses in the L5 dorsal horn of rats with below-level pain after SCI. We show that SCI of sufficient severity induces allodynic-like behavior and robust microglial activation 10 segments below the injury site. Importantly, we are the first to describe the contusion-induced inflammatory microenvironment at L5 where these microglia reside. Elevation of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 occurred in the lumbar spinal

Conclusion

This study identifies remote microglial and p38 activations as predictors of below-level allodynia after SCI. It also specifies the onset and time course of pro-inflammatory cytokine cascades that may be responsible for perpetuating this allodynia. As pain is a multifaceted perception, it is unlikely that microglial modulation is the sole mediator of allodynia. Indeed, several other mechanisms of below-level pain have been elucidated by this lab and others (Christensen et al., 1996b,

Acknowledgments

We express our sincerest thanks to Patricia Walters, A. Todd Lash, Wenmin Lai, Ming Wang, Anne D. Kloos, Zachary Kloos, Richa B. Tripathi, and Emily Hoschouer of The Ohio State University's Center for Brain and Spinal Cord Repair (CBSCR) for their assistance with animal care, surgery and behavioral procedures. Support for this work was contributed by NINDS #NS43798 (DMB), NINDS #NS37846 (PGP), F31 # NS058138 (MRD) and P30-NS045758.

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      Following SCI, multiple pathophysiological changes have been shown to contribute to the development of chronic neuropathic pain (Hulsebosch et al., 2009). These include hyperexcitability of spinal cord dorsal horn neurons (Carlton et al., 2009; Gwak and Hulsebosch, 2011; Hulsebosch et al., 2009), hyperexcitability and heightened activity of primary dorsal root ganglion neurons (Bedi et al., 2012; Bedi et al., 2010; Gwak and Hulsebosch, 2009, 2011; Walters, 2012; Yang et al., 2014), sprouting of primary afferent fibers into the dorsal horn (Krenz and Weaver, 1998; Ondarza et al., 2003; Zinck et al., 2007), and glial activation (Cao and Zhang, 2008; Detloff et al., 2008; Gwak and Hulsebosch, 2009; Hulsebosch, 2008; Tan et al., 2009; Watson et al., 2014). The most immediate effect of SCI is loss of neurons at the lesion epicenter due to mechanical tissue damage; however, it is not clear how lesion size or location affects the development of pain.

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