Camilla I. Svensson

Activation of p38 mitogen‐activated protein kinase in spinal microglia is a critical link in inflammation‐induced spinal pain processing

We examined the effect of p38 mitogen‐activated protein kinase (MAPK) inhibitors in models of nociception and correlated this effect with localization and expression levels of p38 MAPK in spinal cord. There was a rapid increase in phosphorylated p38 MAPK in spinal cord following intrathecal administration of substance P or intradermal injection of formalin. Immuncytochemisty revealed that phosphorylated p38 MAPK‐immunoreactive cells were predominantly present in laminae I–IV of the dorsal horn. Double‐staining with markers for neurons, microglia, astrocytes and oligodendrocytes unexpectedly revealed co‐localization with microglia but not with neurons or other glia. Pretreatment with p38 MAPK inhibitors (SB20358 or SD‐282) had no effect on acute thermal thresholds. However, they attenuated hyperalgesia in several nociceptive models associated with spinal sensitization including direct spinal activation (intrathecal substance P) and peripheral tissue inflammation (intraplantar formalin or carrageenan). Spinal sensitization, manifested by enhanced expression of cyclo‐oxygenase‐2 and inflammation‐induced appearance of Fos‐positive neurons, was blocked by pretreatment, but not post‐treatment, with p38 MAPK inhibitors. Taken together, these results indicate that spinal p38 MAPK is involved in inflammation‐induced pain and that activated spinal microglia play a direct role in spinal nociceptive processing.

Olfactory exposure to males, including men, causes stress and related analgesia in rodents

We found that exposure of mice and rats to male but not female experimenters produces pain inhibition. Male-related stimuli induced a robust physiological stress response that results in stress-induced analgesia. This effect could be replicated with T-shirts worn by men, bedding material from gonadally intact and unfamiliar male mammals, and presentation of compounds secreted from the human axilla. Experimenter sex can thus affect apparent baseline responses in behavioral testing.

Intrathecal minocycline attenuates peripheral inflammation-induced hyperalgesia by inhibiting p38 MAPK in spinal microglia.

Activation of p38 mitogen‐activated protein kinase (p38) in spinal microglia is implicated in spinal nociceptive processing. Minocycline, a tetracycline derivative, displays selective inhibition of microglial activation, a function that is distinct from its antibiotic activity. In the present study we examined antinociceptive effects of intrathecal (IT) administration of minocycline in experimental models of inflammation‐evoked hyperalgesia in addition to the effect of minocycline on stimulation‐induced activation of p38 in spinal microglia. Intrathecal minocycline produced a dose‐dependent reduction of formalin‐evoked second‐phase flinching behaviour in rats, and prevented thermal hyperalgesia induced by carrageenan injection into the paw. In contrast, systemic delivery (intraperitoneally) of minocycline inhibited the first but not the second phase of formalin‐induced flinching, and it had no effect on carrageenan‐induced hyperalgesia. Centrally mediated hyperalgesia induced by IT delivery of N‐methyl‐d‐aspartate was completely blocked by IT minocycline. An increase in phosphorylation (activation) of p38 (P‐p38) was observed in the dorsal spinal cord after carrageenan paw injection, assessed by both Western blotting and immunohistochemistry. The increased P‐p38 immunoreactivity was seen primarily in microglia but also in a small population of neurons. Minocycline, at the IT dose that blocked carrageenan‐induced hyperalgesia, also attenuated the increased P‐p38 in microglia. In addition, minocycline suppressed lipopolysaccharide‐evoked P‐p38 in cultured spinal microglial cells. Taken together, these findings show that minocycline given IT produces a potent and consistent antinociception in models of tissue injury and inflammation‐evoked pain, and they provide strong support for the idea that this effect is mediated by direct inhibition of spinal microglia and subsequent activation of p38 in these cells.

Selective increase of tumour necrosis factor-alpha in injured and spared myelinated primary afferents after chronic constrictive injury of rat sciatic nerve

Chronic constriction of the sciatic nerve, leading to a hyperalgesic state, results in a partial lesion wherein some axons are injured and others remain intact. Here we sought to characterize reactive changes which occur in DRG cell bodies of injured and uninjured axons projecting to skin and muscle. Using immunohistochemistry combined with flurorogold and fluororuby retrograde labelling to define DRG cell bodies associated with injured and uninjured axons, we analysed the DRG immunoreactivity (IR) for tumour necrosis factor‐alpha (TNF), interleukin‐10 (IL‐10), the sensory neuron‐specific channel vanilloid receptor 1 (VR1), isolectin B4 (IB4) and calcitonin‐gene‐related peptide (CGRP) 4 days after a unilateral chronic constriction injury (CCI) of the rat sciatic nerve. TNF IR was predominantly localized in neuronal DRG cells. In DRG with an intact nerve, TNF IR was present in 45%, IL‐10 IR in 46%, VR1 IR in 44%, IB4 IR in 51% and CGRP IR in 40% of all neuronal profiles. Four days after CCI, TNF IR was increased in medium‐sized neurons, whereas IR for IL‐10, VR1 and IB4, predominantly present in small neurons, was reduced. Importantly, not only injured but also adjacent spared neurons contributed markedly to increased TNF IR. Neurons projecting to both muscle and skin displayed upregulated TNF IR after CCI. TNF in medium‐sized neurons colocalized with neurofilament and trkB, but not with IB4, trkA or RET, suggesting a selective phenotypic switch in presumably low‐threshold myelinated primary afferents. Spared myelinated fibres with intact sensory functions but upregulated TNF expression may contribute to behavioural changes observed after nerve injury.

Lipoxins and aspirin-triggered lipoxin inhibit inflammatory pain processing

Inflammatory conditions can lead to debilitating and persistent pain. This hyperalgesia reflects sensitization of peripheral terminals and facilitation of pain signaling at the spinal level. Studies of peripheral systems show that tissue injury triggers not only inflammation but also a well-orchestrated series of events that leads to reversal of the inflammatory state. In this regard, lipoxins represent a unique class of lipid mediators that promote resolution of inflammation. The antiinflammatory role of peripheral lipoxins raises the hypothesis that similar neuraxial systems may also down-regulate injury-induced spinal facilitation of pain processing. We report that the lipoxin A4 receptor is expressed on spinal astrocytes both in vivo and in vitro and that spinal delivery of lipoxin A4, as well as stable analogues, attenuates inflammation-induced pain. Furthermore, activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase in astrocytes, which has been indicated to play an important role in spinal pain processing, was attenuated in the presence of lipoxins. This linkage opens the possibility that lipoxins regulate spinal nociceptive processing though their actions upon astrocytic activation. Targeting mechanisms that counterregulate the spinal consequences of persistent peripheral inflammation provide a novel endogenous mechanism by which chronic pain may be controlled.

The Rheb-mTOR pathway is upregulated in reactive astrocytes of the injured spinal cord

Astrocytes in the CNS respond to tissue damage by becoming reactive. They migrate, undergo hypertrophy, and form a glial scar that inhibits axon regeneration. Therefore, limiting astrocytic responses represents a potential therapeutic strategy to improve functional recovery. It was recently shown that the epidermal growth factor (EGF) receptor is upregulated in astrocytes after injury and promotes their transformation into reactive astrocytes. Furthermore, EGF receptor inhibitors were shown to enhance axon regeneration in the injured optic nerve and promote recovery after spinal cord injury. However, the signaling pathways involved were not elucidated. Here we show that in cultures of adult spinal cord astrocytes EGF activates the mTOR pathway, a key regulator of astrocyte physiology. This occurs through Akt-mediated phosphorylation of the GTPase-activating protein Tuberin, which inhibits Tuberins ability to inactivate the small GTPase Rheb. Indeed, we found that Rheb is required for EGF-dependent mTOR activation in spinal cord astrocytes, whereas the Ras–MAP kinase pathway does not appear to be involved. Moreover, astrocyte growth and EGF-dependent chemoattraction were inhibited by the mTOR-selective drug rapamycin. We also detected elevated levels of activated EGF receptor and mTOR signaling in reactive astrocytes in vivo in an ischemic model of spinal cord injury. Furthermore, increased Rheb expression likely contributes to mTOR activation in the injured spinal cord. Interestingly, injured rats treated with rapamycin showed reduced signs of reactive gliosis, suggesting that rapamycin could be used to harness astrocytic responses in the damaged nervous system to promote an environment more permissive to axon regeneration.

Spinal p38β isoform mediates tissue injury‐induced hyperalgesia and spinal sensitization

Antagonist studies show that spinal p38 mitogen‐activated protein kinase plays a crucial role in spinal sensitization. However, there are two p38 isoforms found in spinal cord and the relative contribution of these two to hyperalgesia is not known. Here we demonstrate that the isoforms are distinctly expressed in spinal dorsal horn: p38α in neurons and p38β in microglia. In lieu of isoform selective inhibitors, we examined the functional role of these two individual isoforms in nociception by using intrathecal isoform‐specific antisense oligonucleotides to selectively block the expression of the respective isoform. In these rats, down‐regulation of spinal p38β, but not p38α, prevented nocifensive flinching evoked by intraplantar injection of formalin and hyperalgesia induced by activation of spinal neurokinin‐1 receptors through intrathecal injection of substance P. Both intraplantar formalin and intrathecal substance P produced an increase in spinal p38 phosphorylation and this phosphorylation (activation) was prevented when spinal p38β, but not p38α, was down‐regulated. Thus, spinal p38β, probably in microglia, plays a significant role in spinal nociceptive processing and represents a potential target for pain therapy.

Peripheral inflammation induces tumor necrosis factor dependent AMPA receptor trafficking and Akt phosphorylation in spinal cord in addition to pain behavior

&NA; In the present study, intraplantar carrageenan induced increased mechanical allodynia, phosphorylation of PKB/Akt and GluR1 ser 845 (PKA site) as well as GluR1, but not GluR2 movement into neuronal membranes. This change in membrane GluR1/GluR2 ratio is indicative of Ca2+ permeable AMPA receptor insertion. Pain behavior was reduced and biochemical changes blocked by spinal pretreatment, but not post‐treatment, with a tumor necrosis factor (TNF) antagonist, Etanercept (100 &mgr;g). Pain behavior was also reduced by spinal inhibition of phosphatidylinositol 3‐kinase (PI‐3K) (wortmannin; 1 and 5 &mgr;g) and LY294002; 50 and 100 &mgr;g) and Akt (Akt inhibitor IV; 3 &mgr;g). Phosphorylated Akt was found exclusively in neurons in grey matter and in oligodendrocytes in white matter. Interestingly, this increase was seen first in superficial dorsal horn and &agr;‐motor neurons (peak 45 min) and later (peak 2 h post‐injection) in deep dorsal horn neurons. Akt and GluR1 phosphorylation, AMPA receptor trafficking and mechanical allodynia were all TNF dependent. Whether phosphorylation of Akt and of GluR1 are in series or in parallel or upstream of pain behavior remains to be determined. Certainly, TNF‐mediated GluR1 trafficking appears to play a major role in inflammatory pain and TNF‐mediated effects such as these could represent a path by which glia contribute to neuronal sensitization (spinal LTP) and pathological pain.

Spinal blockade of TNF blocks spinal nerve ligation-induced increases in spinal P-p38

Spinal nerve ligation (SNL) results in a profound long lasting allodynia and increases in phosphorylated p38 in dorsal root ganglia (DRG) neurons and spinal cord microglia. We have previously shown that systemic etanercept, a tumor necrosis factor (TNF) antagonist, reduced allodynia by 42% and blocked SNL-induced increases in P-p38 levels in the L5 and L6 DRG, but not in the ipsilateral lumbar spinal cord. The present experiments demonstrated that intrathecal etanercept (100 microg) prevents SNL-induced increased levels of spinal P-p38. Pretreatment, but not posttreatment, with intrathecal etanercept (100 microg), given every third day, reduced mechanical allodynia by 50%. This therapeutic benefit was maintained for at least 7 days after cessation of treatment. Combined systemic and intrathecal administration of etanercept was no more effective than intrathecal treatment alone. These data imply that TNF provides the trigger for phosphorylation of p38 in both DRG neurons and spinal microglia.

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss

Objectives Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. Methods Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. Results Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. Conclusions We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.