Bone morphogenetic protein-2-mediated pain and inflammation in a rat model of posterolateral arthrodesis.

BACKGROUND: Bone morphogenetic protein-2 (BMP-2) is a pleiotropic, secreted molecule with diverse effects. The potent ability of BMP-2 to stimulate bone growth prompted its widespread clinical use for arthrodesis (spine fusion). However, elevated post-operative pain in patients treated with BMP-2 has been increasingly reported. Determining whether BMP-2 induces pain directly or whether it induces neuroinflammation, which could lower the threshold for pain, is important for developing therapeutic interventions. We therefore modeled the clinical use of BMP-2 for posterior lumbar fusion by implanting absorbable collagen sponges soaked with either recombinant human BMP-2 (rhBMP-2) or vehicle above the L4-L5 transverse processes of rat spine. RESULTS: Using microarray analysis we found that implantation of rhBMP-2-soaked absorbable collagen sponges resulted in altered expression of numerous pro-inflammatory genes in the adjacent dorsal root ganglia (DRG) showing that implantation of rhBMP-2/absorbable collagen sponges triggers potent neuroinflammatory responses in the DRG-2. Interestingly, direct BMP-2 treatment of DRG explants resulted in changes in gene expression that were not specifically pro-inflammatory. Rats implanted with rhBMP-2 in absorbable collagen sponges also exhibited a transient change in thermal and mechanical sensitivity indicating that rhBMP-2 applied to the lumbar spine could increase pain sensitivity. Immunohistochemical analysis indicated macrophage infiltration in the DRG and spinal nerve in rats implanted with rhBMP-2/absorbable collagen sponges or absorbable collagen sponges alone, but not in rats that underwent surgery without implantation of the absorbable collagen sponges suggesting that the sponges contributed to the biological response. Indeed, analysis of DRGs taken from rats implanted with absorbable collagen sponges without rhBMP-2 showed a significant change in gene expression distinct from DRGs from rats undergoing surgery only. CONCLUSIONS: Our data indicate that implantation of rhBMP-2/absorbable collagen sponges on the lumbar spine triggers potent neuroinflammatory responses in the DRG. Importantly, however, these BMP-2 effects may be partially mediated through a response to the absorbable collagen sponges.

LPS antagonism of TGF-ß signaling results in prolonged survival and activation of rat primary microglia.

Accumulating evidence indicates that activated microglia contribute to the neuropathology involved in many neurodegenerative diseases and after traumatic injury to the CNS. The cytokine transforming growth factor-beta 1 (TGF-ß1), a potent deactivator of microglia, should have the potential to reduce microglial-mediated neurodegeneration. It is therefore perplexing that high levels of TGF-ß1 are found in conditions where microglia are chronically activated. We hypothesized that TGF-ß1 signaling is suppressed in activated microglia. We therefore activated primary rat microglia with lipopolysaccharide (LPS) and determined the expression of proteins important to TGF-ß1 signaling. We found that LPS treatment decreased the expression of the TGF-ß receptors, TßR1 and TßR2, and reduced protein levels of Smad2, a key mediator of TGF-ß signaling. LPS treatment also antagonized the ability of TGF-ß to suppress expression of pro-inflammatory cytokines and to induce microglial cell death. LPS treatment similarly inhibited the ability of the TGF-ß related cytokine, Activin-A, to down-regulate expression of pro-inflammatory cytokines and to induce microglial cell death. Together, these data suggest that microglial activators may oppose the actions of TGF-ß1, ensuring continued microglial activation and survival that eventually may contribute to the neurodegeneration prevalent in chronic neuroinflammatory conditions.

Ablation of rat TRPV1-expressing Adelta/C-fibers with resiniferatoxin: analysis of withdrawal behaviors, recovery of function and molecular correlates.

BACKGROUND: Ablation of TRPV1-expressing nociceptive fibers with the potent capsaicin analog resiniferatoxin (RTX) results in long lasting pain relief. RTX is particularly adaptable to focal application, and the induced chemical axonopathy leads to analgesia with a duration that is influenced by dose, route of administration, and the rate of fiber regeneration. TRPV1 is expressed in a subpopulation of unmyelinated C- and lightly myelinated Adelta fibers that detect changes in skin temperature at low and high rates of noxious heating, respectively. Here we investigate fiber-type specific behaviors, their time course of recovery and molecular correlates of axon damage and nociception using infrared laser stimuli following an RTX-induced peripheral axonopathy. RESULTS: RTX was injected into rat hind paws (mid-plantar) to produce thermal hypoalgesia. An infrared diode laser was used to stimulate Adelta fibers in the paw with a small-diameter (1.6 mm), high-energy, 100 msec pulse, or C-fibers with a wide-diameter (5 mm), long-duration, low-energy pulse. We monitored behavioral responses to indicate loss and regeneration of fibers. At the site of injection, responses to C-fiber stimuli were significantly attenuated for two weeks after 5 or 50 ng RTX. Responses to Adelta stimuli were significantly attenuated for two weeks at the highest intensity stimulus, and for 5 weeks to a less intense Adelta stimulus. Stimulation on the toe, a site distal to the injection, showed significant attenuation of Adelta responses for 7- 8 weeks after 5 ng, or 9-10 weeks after 50 ng RTX. In contrast, responses to C-fiber stimuli exhibited basically normal responses at 5 weeks after RTX. During the period of fiber loss and recovery, molecular markers for nerve regeneration (ATF3 and galanin) are upregulated in the dorsal root ganglia (DRG) when behavior is maximally attenuated, but markers of nociceptive activity (c-Fos in spinal cord and MCP-1 in DRG), although induced immediately after RTX treatment, returned to normal. CONCLUSION: Behavioral recovery following peripheral RTX treatment is linked to regeneration of TRPV1-expressing Adelta and C-fibers and sustained expression of molecular markers. Infrared laser stimulation is a potentially valuable tool for evaluating the behavioral role of Adelta fibers in pain and pain control.

Cytokine gene expression after total hip arthroplasty: surgical site versus circulating neutrophil response.

BACKGROUND: After surgery, cytokines and chemokines are released at the surgical wound site, which can contribute to postoperative pain, local inflammation, and tissue repair. Multiple cell types are present that can release cytokines/chemokines at the wound site and, thus, the exact cellular source of these molecules is unclear. We sought to better understand the contribution of neutrophils to cytokine/chemokine gene expression at the surgical wound site during the initial postsurgery phase of total hip arthroplasty (THA). METHODS: Hip drain fluid was collected at 24 h postsurgery from six patients undergoing standardized THA. In addition, venous blood was collected presurgery and 24 h postsurgery. Neutrophils were isolated, total RNA extracted, and a biotinylated cRNA probe generated. The probes were hybridized with a cDNA microarray containing approximately 100 oligonucleotide sequences representing various human cytokines/chemokines or receptor genes. Changes in gene expression seen in the microarray were verified by reverse transcription polymerase chain reaction. RESULTS: In the microarray analysis of hip drain neutrophils, interleukin-1 receptor antagonist (IL1RN), interleukin-18 receptor 1 (IL18R1), macrophage migration inhibitory factor (MIF), and macrophage inflammatory protein 3alpha (CCL20) were upregulated, whereas interleukin-8 receptor beta (IL8RB/CXCR2) was consistently downregulated, compared with presurgery blood neutrophils. All of these changes were confirmed by reverse transcription polymerase chain reaction. CONCLUSION: There is a distinct cytokine gene expression profile in neutrophils at the THA surgical wound site at 24 h postsurgery when compared with that found in presurgery circulating neutrophils. Understanding these changes may allow us to knowledgeably manipulate neutrophil activity to reduce postoperative pain and inflammation without impairing wound healing.

Peripheral inflammation increases Scya2 expression in sensory ganglia and cytokine and endothelial related gene expression in inflamed tissue.

The sensation of pain (nociception) is a critical factor in host defense during tissue injury and inflammation and is initiated at the site of injury by activation of primary afferent C-fiber and A- partial differential nerve endings. Inflammation induces tissue alterations that sensitize these nociceptive nerve terminals, contributing to persistent pain. To understand this 'algesic tissue environment' and peripheral nervous signaling to the CNS and immune system, we examined cytokine and endothelial-related gene expression profiles in inflamed rat tissues and corresponding dorsal root ganglia (DRG) by microarray and RT-PCR following hind paw injection of carrageenan. In inflamed tissue, forty-two cytokine and endothelial-related genes exhibited elevated expression. In contrast, in DRG, only Scya2 (chemokine C-C motif ligand 2) mRNA was up-regulated, leading to an increase in its gene product monocyte chemoattractant protein-1. Scya2 mRNA was localized by in situ hybridization-immunocytochemical double-labeling to a subpopulation of vanilloid receptor-1 (transient receptor potential vanilloid subtype 1) containing neurons, and its expression was increased by direct transient receptor potential vanilloid subtype 1 stimulation with the vanilloid agonist resiniferatoxin, indicating sensitivity to nociceptive afferent activity. Our results are consistent with the idea that monocyte chemoattractant protein-1 at the site of peripheral injury and/or in DRG is involved in inflammatory hyperalgesia.

Microglial Activation Results in Inhibition of TGF-ß-Regulated Gene Expression.

Chronic inflammation mediated by persistent microglial activation is associated with the pathogenesis of neurodegenerative diseases. The mechanisms underlying chronic microglial activation are poorly understood. We have previously shown that anti-inflammatory TGF-ß signaling is inhibited in LPS-treated microglia. In this study, we assessed whether different disease-related microglial activators could downregulate TGF-ß induction of gene expression. We examined the effects of amyloid ß (Aß) (1-42)- or heat-killed Listeria monocytogenes (HKLM) on the TGF-ß-regulated gene expression in primary rat microglia. We found that Aß (1-42) oligomers and HKLM, in addition to LPS, suppressed TGF-ß-mediated induction of gene expression in part through reducing expression of TßR1 mRNA encoding the TGF-ß receptor 1 in primary microglia. Aß (1-42) and LPS also prevented induction of TGF-ß-induced genes in primary microglia. Additionally, Aß (1-42) rescued primary microglia from TGF-ß-mediated cell death without increasing cell proliferation. Blockage of NFκB signaling, but not the ERK or IRF3 pathways, inhibited Aß (1-42)- and LPS-mediated reduction of TßR1 mRNA. Finally, LPS and Aß (1-42) induced transient upregulation of mRNAs encoding SnoN and Bambi, inhibitors of TGF-ß signaling. Our data indicate that one mechanism through which activators may prolong microglial stimulation is through direct inhibition of anti-inflammatory signaling. A more detailed understanding of the interaction between inflammatory and anti-inflammatory pathways may reveal potential targets for ameliorating chronic inflammation and hence speed the development of therapeutics to address neurodegenerative diseases.

Nociception and inflammatory hyperalgesia evaluated in rodents using infrared laser stimulation after Trpv1 gene knockout or resiniferatoxin lesion.

TRPV1 is expressed in a subpopulation of myelinated Aδ and unmyelinated C-fibers. TRPV1+ fibers are essential for the transmission of nociceptive thermal stimuli and for the establishment and maintenance of inflammatory hyperalgesia. We have previously shown that high-power, short-duration pulses from an infrared diode laser are capable of predominantly activating cutaneous TRPV1+ Aδ-fibers. Here we show that stimulating either subtype of TRPV1+ fiber in the paw during carrageenan-induced inflammation or following hind-paw incision elicits pronounced hyperalgesic responses, including prolonged paw guarding. The ultrapotent TRPV1 agonist resiniferatoxin (RTX) dose-dependently deactivates TRPV1+ fibers and blocks thermal nociceptive responses in baseline or inflamed conditions. Injecting sufficient doses of RTX peripherally renders animals unresponsive to laser stimulation even at the point of acute thermal skin damage. In contrast, Trpv1-/- mice, which are generally unresponsive to noxious thermal stimuli at lower power settings, exhibit withdrawal responses and inflammation-induced sensitization using high-power, short duration Aδ stimuli. In rats, systemic morphine suppresses paw withdrawal, inflammatory guarding, and hyperalgesia in a dose-dependent fashion using the same Aδ stimuli. The qualitative intensity of Aδ responses, the leftward shift of the stimulus-response curve, the increased guarding behaviors during carrageenan inflammation or after incision, and the reduction of Aδ responses with morphine suggest multiple roles for TRPV1+ Aδ fibers in nociceptive processes and their modulation of pathological pain conditions.

RT-PCR analysis of pain genes: use of gel-based RT-PCR for studying induced and tissue-enriched gene expression.

Frequently, it is important to ascertain whether a molecule that is involved in one model of pain is also involved in other models of pain. Similarly, it may be important to determine whether a molecule involved in nociception in one tissue is also expressed in other tissues and to ascertain the degree of enrichment. Additionally, before initiating a complex set of experiments or purchasing an expensive immunoassay kit, it may be useful to obtain initial supporting evidence to justify the time and money. Is the transcript for the target receptor, protein, or peptide precursor present in, for example, the dorsal root ganglion? And, if present, how abundant is it? Here is where the power of PCR can be applied to obtain a quick but informative answer. In this chapter, we mainly detail the use of gel-based RT-PCR and also provide suggestions on tissue dissection and interpretation of results. The use of gel-based RT-PCR can address many of the questions of abundance or tissue specificity with a minimum of expense and time.

Prolonged analgesic response of cornea to topical resiniferatoxin, a potent TRPV1 agonist.

Analgesics currently available for the treatment of pain following ophthalmic surgery or injury are limited by transient effectiveness and undesirable or adverse side effects. The cornea is primarily innervated by small-diameter C-fiber sensory neurons expressing TRPV1 (transient receptor potential channel, subfamily V, member 1), a sodium/calcium cation channel expressed abundantly by nociceptive neurons and consequently a target for pain control. Resiniferatoxin (RTX), a potent TRPV1 agonist, produces transient analgesia when injected peripherally by inactivating TRPV1-expressing nerve terminals through excessive calcium influx. The aim of the present study was to evaluate topical RTX as a corneal analgesic. In rat cornea, a single application of RTX dose dependently eliminated or reduced the capsaicin eye wipe response for 3-5 days, with normal nociceptive responses returning by 5-7 days. RTX alone produced a brief but intense noxious response, similar to capsaicin, necessitating pretreatment of the cornea with a local anesthetic. Topical lidocaine, applied prior to RTX, blocks acute nociceptive responses to RTX without impairing the subsequent analgesic effect. Importantly, RTX analgesia (a) did not impair epithelial wound healing, (b) left the blink reflex intact and (c) occurred without detectable histological damage to the cornea. Immunohistochemistry showed that loss of CGRP immunoreactivity, a surrogate marker for TRPV1-expressing fibers, extended at least to the corneal-scleral boundary and displayed a progressive return, coincident with the return of capsaicin sensitivity. These data suggest that RTX may be a safe and effective treatment for post-operative or post-injury ophthalmic pain.

Localization of S100A8 and S100A9 expressing neutrophils to spinal cord during peripheral tissue inflammation.

Investigation of hyperalgesia at the spinal transcriptome level indicated that carrageenan-induced inflammation of rat hind paws leads to a rapid but sustained increase in S100A8 and S100A9 expression, two genes implicated in the pathology of numerous inflammatory diseases including rheumatoid arthritis and gout. In situ hybridization revealed that the elevation occurred in neutrophils that migrate to the spinal cord vasculature during peripheral inflammation, not in spinal neurons or glial cells. Immunohistochemical analysis suggests, but does not prove, that these neutrophils abundantly release S100A8 and S100A9. Consistent with this, we detected an increase in ICAM and VCAM, both indicators of endothelial activation, a known trigger for secretion of S100A8 and S100A9. Migration of S100A8- and S100A9-expressing neutrophils to spinal cord is selective, since MCP-1- and CD68-expressing leukocytes do not increase in spinal cord vasculature during hind paw inflammation. Examination of many neutrophil granule mediators in spinal cord indicated that they are not regulated to the same degree as S100A8 and S100A9. Neutrophil migration also occurs in the vasculature of brain and pituitary gland during peripheral inflammation. Together, these findings suggest an interaction between a subpopulation of leukocytes and the CNS during peripheral tissue inflammation, as implied by an apparent release and possible diffusion of S100A8 and S100A9 through the endothelial blood-brain barrier. Although the present findings do not establish the neurophysiological or behavioral relevance of these observations to nociceptive processing, the data raise the possibility that selective populations of leukocytes may communicate the presence of disease or tissue damage from the periphery to cells in the central nervous system.