The effect of site and type of nerve injury on spinal glial activation and neuropathic pain behavior.

A number of rat peripheral neuropathy models have been developed to simulate human neuropathic pain conditions. The current study sought to determine the relative importance of site versus type of peripheral nerve injury in eliciting mechanical allodynia and spinal glial responses. Rats received one of seven different surgical treatments at the L5 spinal level: spinal nerve cryoneurolysis, spinal nerve tight ligation, dorsal root cryoneurolysis, dorsal root tight ligation, dorsal root transection, ventral root tight ligation, or laminectomy/dural incision sham. Foot-lift response frequency to mechanical stimulation of the ipsilateral hindpaw was assessed postlesion on days 1, 3, 5, and 7. L5 spinal cords were retrieved for immunohistochemical analysis of microglial (OX-42) and astrocytic (anti-glial fibrillary acidic protein) responses. Both types of spinal nerve lesion, freeze and tight ligation, produced rapid and profound mechanical allodynia with intense glial responses. Dorsal root lesions also resulted in intense mechanical allodynia; however, glial responses were almost exclusively astrocytic. Ventral root tight ligation and sham provoked no marked behavioral changes and only sporadic glial responses. Direct dorsal horn communication with the dorsal root ganglion was not a crucial factor in the development of mechanical allodynia, since decentralization of the L5 DRG by complete L5 dorsal root lesion produced profound mechanical sensitization. Conversely, microglial activation responses appear to be dependent upon dorsal root ganglion-mediated signals and, contrary to behavioral responses, were robust only when the lesion was made peripheral to the cell body. Astrocytic activation was always observed following axonal injury and reliably coexisted with behavioral responses.

Increase of interleukin-6 mRNA in the spinal cord following peripheral nerve injury in the rat: potential role of IL-6 in neuropathic pain.

Interleukin-6 (IL-6) is a multifunctional cytokine whose actions include modulation of proliferation, differentiation, and maturation of hemapoietic progenitors and other cell lineages; growth regulation of certain carcinoma cell lines; and control of cellular metabolic activities. Initially described in terms of its activities in the immune system and inflammation, accumulating evidence supports an essential role of IL-6 in the development, differentiation, regeneration and degeneration of neurons in the peripheral and central nervous system. We have previously demonstrated that immunoreactive-like IL-6 protein is significantly elevated in the spinal cord in response to peripheral nerve injury that results in neuropathic pain behaviors in the rat. In the current study, our objective was to determine if the source of IL-6 protein was endogenous to the central nervous system by measuring any detectable increases in spinal IL-6 mRNA expression following established mononeuropathy procedures associated with neuropathic pain: spinal nerve cryoneurolysis (SPCN) or spinal nerve tight ligation (SPTL). Using in situ hybridization and a digoxigenin-labeled oligonucleotide, IL-6 mRNA in neurons was significantly elevated at 3 and 7 days post SPCN and 7 days post SPTL in both dorsal and ventral horns. The cellular localization of the IL-6 mRNA expression was predominately neuronal as confirmed by NeuN serial staining. For example, in the SPCN 7 day group, IL-6 mRNA cell profiles in the ipsilateral dorsal horn were significantly different from the normal group (38.7+/-12.8 vs. 4.89+/-1.6, p<0.001). These data demonstrate the central, spinal production of a proinflammatory cytokine in response to a peripheral nerve injury. In addition, these results add to the growing body of literature implicating these immune products, cytokines, as potential neuromodulators/neurotransmitters and provides further evidence for their role in the nociceptive processing which leads to chronic pain.

Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat.

Peripheral nerve injury commonly leads to neuropathic pain states fostered, in part, by neuroimmunologic events. We used two models of neuropathic pain (L5 spinal nerve cryoneurolysis (SPCN) and chronic constriction injury (CCI)) to assess the role of spinal glial activation responses in producing pain behaviors. Scoring of glial responses subjectively encompassed changes in cell morphology, cell density and intensity of immunoreactivity with specific activation markers (OX-42 and anti-glial fibrillary acidic protein (GFAP) for microglia and astrocytes, respectively). Glial responses were compared with tactile sensitivity (mechanical allodynia) at 1, 3 or 10 days following SPCN and with thermal hyperalgesia at 10 days in the CCI group. Neuropathic pain behaviors preceded and did not closely correlate with microglial responses in either model. Perineural application of bupivacaine prior to SPCN prevented spinal microglial responses but not pain behaviors. Spinal astrocytic responses to SPCN were early, robust and not altered by bupivacaine. The current findings support the use of bupivacaine as a tool to suppress microglial activation and challenge the putative role of microglia in initiating or potentiating pain behaviors which result from nerve injury.

Cytokine and growth factor immunohistochemical spinal profiles in two animal models of mononeuropathy.

Nerve injury leads to central neuroimmunologic responses that may be integral to the development and maintenance of chronic neuropathic pain in humans. Recent data have demonstrated that cytokines and growth factors may be strongly implicated in the generation of pain states at both peripheral and central nervous system sites. We utilized immunohistochemical methods to investigate this phenomenon in rat models of neuropathic pain. Specifically, we employed well-characterized models of neuropathy that result in behaviors suggestive of neuropathic pain in humans; a freeze lesion of the sciatic nerve, termed sciatic cryoneurolysis, and a chronic constriction sciatic nerve injury. We used immunohistochemistry to examine spinal localization of the cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and the growth factors, basic fibroblast growth factor (bFGF), and transforming growth factor-beta1 (TGF-beta) at 3, 14, and 35 days following sciatic cryoneurolysis or 6 days following chronic constriction injury as compared with normal, unoperated rats. There was minimal, diffuse cytokine/growth factor staining in lumbar spinal tissue from the normal group. However, cell profile quantification demonstrated increases in lumbar spinal IL-1beta-, TNF-alpha- and TGF-beta-like immunoreactivity (LI) in both mononeuropathy models studied. At 3 days following sciatic cryoneurolysis, intense bFGF LI was present in the ipsilateral dorsal and ventral horn. By 14 days bFGF LI was also observed in contralateral dorsal and ventral horns. In contrast, we found no obvious staining differences in lumbar spinal cord following the chronic constriction injury. This study demonstrated increased specific cytokine and growth factor-like expression in the spinal cord following peripheral nerve injuries. It also showed a differential expression of bFGF in two distinct mononeuropathy models. These results provide further evidence that central cytokine production via a neuroimmune cascade may be involved in the development and maintenance of behaviors that mimic neuropathic pain following nerve injury.

Intrathecal catheterization alone induces neuroimmune activation in the rat.

Intrathecal (i.t.) catheterization in the rat has been used extensively for drug delivery in various experimental paradigms. These indwelling i.t. catheters have been associated with inflammatory processes and tissue reactions external to the spinal cord in numerous clinical and animal studies. The purpose of this study was to determine whether i.t. catheter placement produced glial activation and changes in specific cytokine expression, i.e. neuroimmune activation, within the spinal cord which might cause altered sensory processing. Rats underwent i.t. catheterization or sham surgery and were killed at 3 or 14 days postsurgery (n> or =3 per group). Spinal cord segments were taken at the cervical level, tip of the catheter and distal to the catheter (thoracic levels). Immunohistochemistry was used to examine spinal localization of the cytokines, interleukin (IL)-6, IL-10 and glial activation (OX-42 for microglia and anti-glial fibrillary acidic protein for astrocytes). At 3 and 14 days after i.t. catheterization, there was an elevation in OX-42 and GFAP expression as compared to control (n=3) and sham surgery (n=4) groups. IL-10-like immunoreactivity was significantly increased in both the dorsal and ventral horns 14 days after i.t. placement as compared to the sham and normal groups. Conversely, IL-6-like immunoreactivity was not significantly different from sham or normal groups. These cytokine findings are discussed in the context of a differential role of specific cytokines in the potential generation of pain states or in the production of analgesia. This study demonstrated that i.t. catheterization induces robust neuroimmune activation that manifests as increases in glial markers and specific cytokine expression. This method should be controlled for, or alternate methods used for, drug delivery in nociceptive animal models that require spinally administered agents.