Delay of neuropathic pain sensitization after application of dexamethasone-loaded implant in sciatic nerve-injured rats

Neuroimmune interactions underlying the development of pain sensitization in models of neuropathic pain have been widely studied. In this study, we evaluated the development of allodynia and its reduction associated with peripheral antineuroinflammatory effects induced by a dexamethasone-loaded biodegradable implant. Chronic constriction injury (CCI) of the sciatic nerve was performed in Wistar rats. The electronic von Frey test was applied to assess mechanical allodynia. The dexamethasone-loaded implant was placed perineurally at the moment of CCI or 12 days after surgery. Dorsal root ganglia (DRG; L4-L5) were harvested and nuclear extracts were assayed by Western blot for detection of nuclear factor (NF)-κB p65/RelA translocation. Dexamethasone delivered from the implant delayed the development of allodynia for approximately three weeks in CCI rats when the implantation was performed at day 0, but allodynia was not reversed when the implantation was performed at day 12. NF-κB was activated in CCI rat DRG compared with naïve or sham animals (day 15), and dexamethasone implant inhibited p65/ RelA translocation in CCI rats compared with control. This study demonstrated that the dexamethasoneloaded implant suppresses allodynia development and peripheral neuroinflammation. This device can reduce the potential side effects associated with oral anti-inflammatory drugs

Annexin A1 and the Resolution of Inflammation: Modulation of Neutrophil Recruitment, Apoptosis, and Clearance

Neutrophils (also named polymorphonuclear leukocytes or PMN) are essential components of the immune system, rapidly recruited to sites of inflammation, providing the first line of defense against invading pathogens. Since neutrophils can also cause tissue damage, their fine-tuned regulation at the inflammatory site is required for proper resolution of inflammation. Annexin A1 (AnxA1), also known as lipocortin-1, is an endogenous glucocorticoid-regulated protein, which is able to counterregulate the inflammatory events restoring homeostasis. AnxA1 and its mimetic peptides inhibit neutrophil tissue accumulation by reducing leukocyte infiltration and activating neutrophil apoptosis. AnxA1 also promotes monocyte recruitment and clearance of apoptotic leukocytes by macrophages. More recently, some evidence has suggested the ability of AnxA1 to induce macrophage reprogramming toward a resolving phenotype, resulting in reduced production of proinflammatory cytokines and increased release of immunosuppressive and proresolving molecules. The combination of these mechanisms results in an effective resolution of inflammation, pointing to AnxA1 as a promising tool for the development of new therapeutic strategies to treat inflammatory diseases

Switching Off Key Signaling Survival Molecules to Switch On the Resolution of Inflammation

Inflammation is a physiological response of the immune system to injury or infection but may become chronic. In general, inflammation is self-limiting and resolves by activating a termination program named resolution of inflammation. It has been argued that unresolved inflammation may be the basis of a variety of chronic inflammatory diseases. Resolution of inflammation is an active process that is fine-tuned by the production of proresolving mediators and the shutdown of intracellular signaling molecules associated with cytokine production and leukocyte survival. Apoptosis of leukocytes (especially granulocytes) is a key element in the resolution of inflammation and several signaling molecules are thought to be involved in this process. Here, we explore key signaling molecules and some mediators that are crucial regulators of leukocyte survival in vivo and that may be targeted for therapeutic purposes in the context of chronic inflammatory diseases

Phosphatidylinositol 3-Kinase γ Is Required for the Development of Experimental Cerebral Malaria

<div><p>Experimental cerebral malaria (ECM) is characterized by a strong immune response, with leukocyte recruitment, blood-brain barrier breakdown and hemorrhage in the central nervous system. Phosphatidylinositol 3-kinase γ (PI3Kγ) is central in signaling diverse cellular functions. Using PI3Kγ-deficient mice (PI3Kγ^-/-) and a specific PI3Kγ inhibitor, we investigated the relevance of PI3Kγ for the outcome and the neuroinflammatory process triggered by <i>Plasmodium berghei</i> ANKA (PbA) infection. Infected PI3Kγ^-/- mice had greater survival despite similar parasitemia levels in comparison with infected wild type mice. Histopathological analysis demonstrated reduced hemorrhage, leukocyte accumulation and vascular obstruction in the brain of infected PI3Kγ^-/- mice. PI3Kγ deficiency also presented lower microglial activation (Iba-1+ reactive microglia) and T cell cytotoxicity (Granzyme B expression) in the brain. Additionally, on day 6 post-infection, CD3⁺CD8⁺ T cells were significantly reduced in the brain of infected PI3Kγ^-/- mice when compared to infected wild type mice. Furthermore, expression of CD44 in CD8+ T cell population in the brain tissue and levels of phospho-IkB-α in the whole brain were also markedly lower in infected PI3Kγ^-/- mice when compared with infected wild type mice. Finally, AS605240, a specific PI3Kγ inhibitor, significantly delayed lethality in infected wild type mice. In brief, our results indicate a pivotal role for PI3Kγ in the pathogenesis of ECM.</p></div

Treatment with a PI3Kγ inhibitor delayed mortality in WT PbA-infected mice.

<p>The selective inhibitor of PI3Kγ, AS605240, was given orally, once a day, at the dose of 30 mg/kg, from day 3 until day 6 p.i. (n = 5). Untreated infected animals (n = 8) received drug vehicle in the same therapeutic scheme. Percentage of survival in PbA-infected mice <b>(A)</b>, where solid lines denote vehicle-treated mice and dashed lines, AS605240-treated mice. Parasitemia levels on day 6 p.i., without differences between infected groups <b>(B)</b>. Significant differences were indicated by *p<0.05 and parasitemia expressed as mean ± SD.</p

Increased resistance in PI3Kγ^-/- mice infected with PbA.

<p>Percentage of survival in PbA-infected mice <b>(A)</b>. C57BL/6 WT (solid lines, n = 5) and PI3Kγ^-/- mice (dashed lines, n = 6). The survival curve of PI3Kγ^-/- was significantly different of WT, with *p<0.05. Percentage of ECM incidence in infected mice <b>(B)</b> based on typical neurological symptoms of ECM, with *p<0.05 between groups. Parasitemia levels <b>(C)</b> from day 3 p.i. up to day 12 p.i., without differences between infected groups. Parasitemia was expressed as mean ± SD. Representative results from 2 independent experiments.</p

Lung pathology induced by PbA infection seems to be independent of PI3Kγ.

<p>Representative photomicrographs of hematoxylin/eosin (HE)-stained lung sections from uninfected control mice <b>(A),</b> or PbA-infected C57BL/6 <b>(B)</b> and PI3Kγ^-/-<b>(C)</b> mice on day 6 p.i. Normal histological section of lung parenchyma with standard architecture from uninfected control <b>(A)</b>. Lung sections from a PbA-infected C57BL/6 and PI3Kγ^-/- animal exhibiting slight interstitial edema, thickening of alveolar septae and a reduction of alveolar size <b>(B, C)</b>. Semi-quantification score for histopathological changes in the lung of PbA-infected mice showed no significant difference between C57BL/6 and PI3Kγ^-/- infected mice (n = 5 mice for each group) <b>(D)</b>. Original magnification: A-D: x200. Sections were captured with a digital camera (Optronics DEI-470) connected to a microscope (Olympus IX70).</p

Infected PI3Kγ^-/- mice present mild histopathological changes in brain.

<p>Representative photomicrographs of hematoxylin/eosin (HE)-stained brain sections from uninfected control mice <b>(A, D),</b> or PbA-infected C57BL/6 <b>(B, E)</b> and PI3Kγ^-/-<b>(C, F)</b> mice on day 6 p.i. Normal histological appearance of cerebrum <b>(A)</b> and brainstem <b>(D)</b> from uninfected control mice. Brain sections from a PbA-infected wild-type animal exhibiting intravascular leukocyte aggregates (asterisks) in cerebrum <b>(B)</b> and hemorrhage (arrow head) in brainstem (E). Note vascular plugging (asterisk) in the deep cerebral cortex <b>(C)</b> and small hemorrhagic foci (arrow head) in the brainstem <b>(F)</b> from PbA-infected PI3Kγ^-/- animal. The severity of microvascular obstruction and cerebellar hemorrhagic areas (μm²/field) in infected mice demonstrated a significant reduction in brain alterations in PI3Kγ^-/- mice <b>(J)</b>. Immunohistochemical staining for anti-Iba-1 showing no reactive microglia (arrow) in non-infected WT mouse brain <b>(G)</b>. Microglial cells (arrows) displaying morphological changes (thickening of the microglial processes) and immunoreactivity for Iba-1 in the cerebrum of PbA-infected wild-type animal (H) and PI3Kγ^-/- mouse (I). Quantification of reactive <b>microglia (K)</b> confirmed lower number in brain sections of infected-PI3Kγ^-/- mice when compared with PbA-infected WT animal. Significant differences were indicated by *p<0.05 (n = 5 mice per group). Original magnification: A-I: x200. Sections were captured with a digital camera (Optronics DEI-470) connected to a microscope (Olympus IX70).</p

Reduced CD8⁺ T cell accumulation, CD8+ T-cell cytotoxicity and activation in the brain of PbA-infected PI3Kγ^-/- mice.

<p>Flow cytometric analyses of brain-sequestered leukocytes from PbA-infected WT and PI3Kγ^-/- mice on day 6 p.i. Absolute numbers of CD3⁺CD8⁺ T cells, expressed as total number per brain <b>(A)</b>. Percentage of Granzyme B⁺ (GrzB⁺) cells in CD8⁺ T population <b>(B)</b> and the expression of CD44 in CD8⁺ T cells <b>(C)</b> presented as mean fluorescence intensity (MFI). Results are expressed as mean ± SD and are representative of 2 independent experiments (n = 4 samples per group, with each sample representing a pool of 2 mice brains). Significant difference between infected groups was indicated by *p<0.05 or **p<0.01.</p