Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat
Introduction
Neuropathic pain, or chronic pain due to nerve injury, is both prevalent and resistant to current pharmacological and non-pharmacological therapies (Arner and Meyerson, 1988). To investigate the etiology of neuropathic pain in humans, our laboratory developed and characterized two rat neuropathy models termed sciatic cryoneurolysis (SCN) and spinal nerve cryoneurolysis (SPCN) (DeLeo et al., 1994; Colburn et al., 1996). The focal peripheral nerve lesion is produced by exposure and freezing of the sciatic nerve (SCN) or the more proximal L5 spinal nerve (SPCN) resulting in a delayed but sustained mechanical allodynia (increased sensitivity to a non-noxious stimulus like touch) following SCN (Willenbring et al., 1994) and immediate and sustained mechanical allodynia and thermal hyperalgesia (increased sensitivity to a noxious, heat stimulus) following SPCN (Colburn et al., 1996). These models have proven ideal for the study of neuropathic pain due to the resultant predictable and robust pain behaviors similar to neuropathic pain experienced clinically.
In order to better understand the pathophysiology associated with differential nerve injury and chronic pain, we compared our regenerative permissive freeze models with another well-established neuropathic pain model, chronic constriction injury (CCI) in the rat (Bennett and Xie, 1988). This injury is produced by loose ligation of the sciatic nerve using chromic gut resulting in an immediate, but not sustained thermal hyperalgesia and mechanical allodynia. Unlike the complete, albeit temporary, nerve injury that is produced by a freeze lesion (Wagner et al., 1995), CCI spares many of the unmyelinated fibers at the lesion site (Carlton et al., 1991). The chemical toxicity of chromic gut (as opposed to silk suture) is required for the development of abnormal behaviors observed following CCI (Maves et al., 1993) suggesting a local, inflammatory neuritis in the etiology of neuropathic pain in this model. Although it is evident that both peripheral and central pathologies are involved in the pathogenesis of chronic neuropathic pain, the ongoing contribution of each is not yet understood. The benefits of investigating differential peripheral nerve injury models include the ability to discern specific peripheral nerve and spinal contributions in the generation and maintenance of neuropathic pain.
Using the neurolysis models and the chronic constriction injury model, we found evidence that immune cell activation and cytokine products of immune cells contribute to the generation of chronic pain states (DeLeo et al., 1996, DeLeo et al., 1997; DeLeo and Colburn, 1996). Our working hypothesis is that nerve injury leads to central neuroimmunologic responses such as cytokine-mediated glial activation or glial-mediated cytokine expression which act to propagate neuropathic pain behaviors either directly or through induced expression or release of common pain mediators such as glutamate and nitric oxide (Hopkins and Rothwell, 1995).
Microglia, members of the monocyte/macrophage family, perform a variety of immune functions and may be part of the neuropathic pain process. Pathological stimuli can provoke a graded transformation of microglia from a highly ramified resting surveillance state ultimately to a phagocytic macrophage. Microglial activation involves a stereotypic pattern of cellular responses, that include proliferation, recruitment to the site of injury, increased expression of immunomolecules (e.g. surface antigens such as CD11b/CD18 and MHC Class II). Following activation, microglia undergo further functional changes including the expression and release of cytotoxic and/or inflammatory mediators, including IL-1β, IL-6, TNF-α, proteases, and reactive oxygen intermediates including nitric oxide (Woodroofe et al., 1991; Streit and Kincaid-Colton, 1995). Interleukin-1β and TNF-α have been associated with generalized hyperalgesia observed following lipopolysaccharide (LPS) administration (Watkins et al., 1995) while nitric oxide is also known to enhance pain transmission (Meller and Gebhart, 1993).
We have recently demonstrated that OX-42 immunoreactivity (ir) and the proinflammatory cytokines, IL-1β, IL-6, TNF-α are elevated in the rat spinal cord following peripheral nerve injury in a time course that correlates with pain behaviors (DeLeo and Colburn, 1996; DeLeo et al., 1997) and that exogenous application of rhIL-6 intrathecally provoked mechanical hyperalgesia in normal rats (DeLeo et al., 1996). Once thought of as merely a physical support system for neurons, glial cells have recently come under intense scrutiny as key neuromodulatory, neurotrophic and neuroimmune elements in the CNS. Since both microglia and astrocytes produce cytokines that are known to have a role in sensitization of the spinal cord (Meller et al., 1994), it is reasonable to suspect that glial activation may play a role in nociceptive processing. In addition to neuromodulation, glia also perform roles in synaptic remodeling and provide neurotrophic support for regenerating neurons. Thus, glia appear to be intimately involved in two primary correlates of chronic neuropathic pain states, namely spinal hypersensitization and synaptic remodeling.
The current study was designed to: (1) Immunocytochemically and morphologically characterize the time course of microglial and astrocytic responses using glial cell markers (OX-42, GFAP) following SPCN and CCI in the rat; (2) to compare the timing and degree of glial responses with pain behavior onset and severity; (3) to assess the effect of perineural local anesthetics in: (a) preventing the development of neuropathic pain behaviors after SPCN and (b) attenuating glial activation responses in the spinal cord.
Section snippets
Animals
All experiments were performed using 225–350 g male Holtzman-strain Sprague–Dawley rats housed under USDA and AAALAC-approved conditions with 12–12 h light–dark cycle and free access to food and water. All experimental procedures were approved by the Dartmouth College and National Institutes of Health Institutional Animal Care and Use Committees.
Spinal nerve cryoneurolysis (SPCN)
Surgery was performed under inhalation anesthesia using halothane in 100% O2, induced at 4% and maintained at 2%. Each SPCN lesioned rat sustained a
Microglial response to SPCN
Profound microglial responses were observed ipsilaterally in the spinal dorsal and ventral horns in rats following the SPCN injury. Naive rats exhibited no overt signs of microglial activation, while sporadic microglial responses to surgery were observed at 1 day post-SPCN and in several sham animals (Table 1). A single rat in the 3 day post-sham group (Table 1) displayed a moderate generalized spinal microglial response which may correlate with the stress of a difficult anesthetic induction or
Discussion
Microglia rapidly and reliably become activated following injury to the CNS. This activation responses takes the form of expression of Major Histocompatibility Complex (MHC) class I and II antigens, inflammatory cytokines, chemokines and inducible nitric oxide synthase (iNOS), as well as, morphological changes (Perry et al., 1993; Kreutzberg, 1996). Microglial response is evident whether the insult takes the form of direct trauma, ischemic injury, infection, or is mediated locally by endogenous
Acknowledgements
This work was funded under National Institute of Drug Abuse grants DA05731-F31 (RWC) and DA10042-01(JAD). We thank Dr. G.J. Bennett for providing CCI rat spinal cord tissue.
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