José Miguel Vela

Therapeutic Action of Cannabinoids in a Murine Model of Multiple Sclerosis

Theilers virus infection of the CNS induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Cannabinoids may act as immunosuppressive compounds that have shown therapeutic potential in chronic inflammatory disorders. Using the Theilers murine encephalomyelitis virus model, we report here that treatment with the synthetic cannabinoids WIN 55,212–2, ACEA, and JWH-015 during established disease significantly improved the neurological deficits in a long-lasting way. At a histological level, cannabinoids reduced microglial activation, abrogated major histocompatibility complex class II antigen expression, and decreased the number of CD4+ infiltrating T cells in the spinal cord. Both recovery of motor function and diminution of inflammation paralleled extensive remyelination. Overall, the data presented may have potential therapeutic implications in demyelinating pathologies such as MS; in particular, the possible involvement of cannabinoid receptor CB2 would enable nonpsychoactive therapy suitable for long-term use.

Prenatal Immune Challenge Disrupts Sensorimotor Gating in Adult Rats: Implications for the Etiopathogenesis of Schizophrenia

Increasing evidence associates schizophrenia with prenatal exposure to infection. Impaired ability to “gate out” sensory and cognitive information is considered to be a central feature of schizophrenia and is manifested, among others, in disrupted prepulse inhibition (PPI) of the acoustic startle reflex. We analyzed the effect of a prenatal immune challenge— peripheral administration of bacterial endotoxin lipopolysaccharide (LPS) to pregnant female rats—upon PPI and immune function in adult offspring. Prenatal LPS treatment disrupted PPI which was reversed by antipsychotics. Serum levels of interleukin-2 and interleukin-6 were increased. In addition, histopathological features in brain areas related with PPI circuitry were observed. These results illustrate the critical influence of prenatal immune events upon adult CNS functioning in association with the putative role of the immune system in the etiopathogenesis of schizophrenia.

LPS/IFN-γ cytotoxicity in oligodendroglial cells: Role of nitric oxide and protection by the anti-inflammatory cytokine IL-10

Proinflammatory mediators have been implicated in demyelinating disorders, including multiple sclerosis, whereas it has been proposed that the anti‐inflammatory cytokines interleukin‐ (IL‐) 4 and IL‐10 participate in disease recovery. The present study analysed the effect of interferon‐γ (IFN‐γ) and bacterial endotoxin (lipopolysaccharide, LPS) on proliferation and survival of progenitors and differentiated oligodendrocytes. We also investigated the presence of receptors for IL‐4 and IL‐10 in oligodendroglial cells and explored a possible protective action of IL‐4 and IL‐10 in cultures following LPS/IFN‐γ. Finally, the role of endogenous nitric oxide (NO) on cell viability and the modulatory action of IL‐4 and IL‐10 on inducible nitric oxide synthase (iNOS) expression were also analysed. We report that LPS and/or IFN‐γ reduced proliferation and viability of oligodendroglial cells. Cell death, presumably by apoptosis as evidence by TUNEL and Annexin V binding, was observed following LPS/IFN‐γ, progenitors being more sensitive than differentiated cells. At both developmental stages, LPS/IFN‐γ‐treated cultures expressed iNOS protein and released micromolar concentrations of NO. In progenitors, LPS/IFN‐γ‐mediated cell damage was partially dependent on endogenous NO production, whereas NO was fundamental for cytotoxicity of differentiated oligodendrocytes. Both cell types expressed mRNA for IL‐4 and IL‐10 receptors and expression of IL‐10 receptors at the protein level was also demonstrated. Treatment with either cytokine inhibited the expression of iNOS resulting from the proinflammatory stimulation. IL‐10 was more effective than IL‐4 in suppressing iNOS expression and, interestingly, IL‐10 conferred protection against oligodendroglial death evoked by LPS/IFN‐γ. Our data raise the question of whether IL‐10 may play a protective role in demyelinating diseases, not only downregulating the function of inflammatory cells but also promoting survival of progenitors and differentiated oligodendrocytes.

Interleukin-1 Regulates Proliferation and Differentiation of Oligodendrocyte Progenitor Cells

Interleukin-1 (IL-1) is a pleiotropic cytokine expressed during normal CNS development and in inflammatory demyelinating diseases, but remarkably little is known about its effect on oligodendroglial cells. In this study we explored the role of IL-1beta in oligodendrocyte progenitors and differentiated oligodendrocytes. The effects of IL-1beta were compared to those of IL-1 receptor antagonist, the specific inhibitor of IL-1 activity, since progenitors and differentiated oligodendrocytes produce IL-1beta and express IL-1 receptors. Unlike other proinflammatory cytokines (TNFalpha and IFNgamma), IL-1beta was not toxic for oligodendrocyte lineage cells. However, this cytokine inhibited proliferation of oligodendrocyte progenitors in the presence of growth factors (PDGF plus bFGF). This was evidenced by a significant decrease in both cells incorporating bromodeoxyuridine (45%) and total cell numbers (57%) after 6 days of treatment. Interestingly, IL-1beta blocked proliferation at the late progenitor/prooligodendrocyte (O4+) stage but did not affect proliferation of early progenitors (A2B5+). Inhibition of proliferation paralleled with promotion of differentiation, as revealed by the increased percentage of R-mab+ cells (6.7-fold). Moreover, when oligodendrocyte progenitors were allowed to differentiate in the absence of growth factors, treatment with IL-1beta promoted maturation to the MBP+ stage (4.2-fold) and survival of differentiating oligodendrocytes (2.1-fold). Regarding intracellular signaling, IL-1beta activated the p38 mitogen-activated protein kinase (MAPK) but not the p42/p44 MAPK and, when combined with growth factors, intensified p38 activation but inhibited the growth-factor-induced p42/p44 activation. IL-1beta also induced a time-dependent inhibition of PFGF-Ralpha gene expression. These results support a role for IL-1beta in promoting mitotic arrest and differentiation of oligodendrocyte progenitors as well as maturation and survival of differentiating oligodendrocytes.

Dynamics of microglia in the developing rat brain

Entrance of mesodermal precursors into the developing CNS is the most well‐accepted origin of microglia. However, the contribution of proliferation and death of recruited microglial precursors to the final microglial cell population remains to be elucidated. To investigate microglial proliferation and apoptosis during development, we combined proliferating cell nuclear antigen (PCNA) immunohistochemistry, in situ detection of nuclear DNA fragmentation (TUNEL), and caspase‐3 immunohistochemistry with tomato lectin histochemistry, a selective microglial marker. The study was carried out in Wistar rats from embryonic day (E) 16 to postnatal day (P) 18 in cerebral cortex, subcortical white matter, and hippocampus. Proliferating microglial cells were found at all ages in the three brain regions and represented a significant fraction of the total microglial cell population. The percentage of microglia expressing PCNA progressively increased from the embryonic period (25–51% at E16) to a maximum at P9, when the great majority of microglia expressed PCNA (92–99%) in all the brain regions analyzed. In spite of the remarkable proliferation and expansion of the microglial population with time, the density of microglia remained quite constant in most brain regions because of the considerable growth of the brain during late prenatal and early postnatal periods. In contrast, apoptosis of microglia was detected only at certain times and was restricted to some ameboid cells in white matter and primitive ramified cells in gray matter, representing a small fraction of the microglial population. Therefore, our results point to proliferation of microglial precursors in the developing brain as a physiological mechanism contributing to the acquisition of the adult microglial cell population. In contrast, microglial apoptosis occurs only locally at certain developmental stages and thus seems less crucial for the establishment of the final density of microglia. J. Comp. Neurol. 458:144–157, 2003.

Sigma-1 receptors regulate activity-induced spinal sensitization and neuropathic pain after peripheral nerve injury

ABSTRACT Sigma‐1 receptor (σ1R) is expressed in key CNS areas involved in nociceptive processing but only limited information is available about its functional role. In the present study we investigated the relevance of σ1R in modulating nerve injury‐evoked pain. For this purpose, wild‐type mice and mice lacking the σ1R gene were exposed to partial sciatic nerve ligation and neuropathic pain‐related behaviors were investigated. To explore underlying mechanisms, spinal processing of repetitive nociceptive stimulation and expression of extracellular signal‐regulated kinase (ERK) were also investigated. Sensitivity to noxious heat of homozygous σ1R knockout mice did not differ from wild‐type mice. Baseline values obtained in σ1R knockout mice before nerve injury in the plantar, cold‐plate and von Frey tests were also indistinguishable from those obtained in wild‐type mice. However, cold and mechanical allodynia did not develop in σ1R null mice exposed to partial sciatic nerve injury. Using isolated spinal cords we found that mice lacking σ1R showed reduced wind‐up responses respect to wild‐type mice, as evidenced by a reduced number of action potentials induced by trains of C‐fiber intensity stimuli. In addition, in contrast to wild‐type mice, σ1R knockout mice did not show increased phosphorylation of ERK in the spinal cord after sciatic nerve injury. Both wind‐up and ERK activation have been related to mechanisms of spinal cord sensitization. Our findings identify σ1R as a constituent of the mechanisms modulating activity‐induced sensitization in pain pathways and point to σ1R as a new potential target for drugs designed to alleviate neuropathic pain.

5-HT7 receptor activation inhibits mechanical hypersensitivity secondary to capsaicin sensitization in mice

ABSTRACT This work aimed to evaluate the potential role of the 5‐HT7 receptor in nociception secondary to a sensitizing stimulus in mice. For this purpose, the effects of relevant ligands (5‐HT7 receptor agonists: AS‐19, MSD‐5a, E‐55888; 5‐HT7 receptor antagonists: SB‐258719, SB‐269970; 5‐HT1A receptor agonist: F‐13640; 5‐HT1A receptor antagonist: WAY‐100635) were assessed on capsaicin‐induced mechanical hypersensitivity, a pain behavior involving hypersensitivity of dorsal horn neurons (central sensitization). For the 5‐HT7 receptor agonists used, binding profile and intrinsic efficacy to stimulate cAMP formation in HEK‐293F cells expressing the human 5‐HT7 receptor were also evaluated. AS‐19 and E‐55888 were selective for 5‐HT7 receptors. E‐55888 was a full agonist whereas AS‐19 and MSD‐5a behaved as partial agonists, with maximal effects corresponding to 77% and 61%, respectively, of the cAMP response evoked by the full agonist 5‐HT. Our in vivo results revealed that systemic administration of 5‐HT7 receptor agonists exerted a clear‐cut dose‐dependent antinociceptive effect that was prevented by 5‐HT7 receptor antagonists, but not by the 5‐HT1A receptor antagonist. The order of efficacy (E‐55888 > AS‐19 > MSD‐5a) matched their in vitro efficacy as 5‐HT7 receptor agonists. Contrary to agonists, a dose‐dependent promotion of mechanical hypersensitivity was observed after administration of 5‐HT7 receptor antagonists, substantiating the involvement of the 5‐HT7 receptor in the control of capsaicin‐induced mechanical hypersensitivity. These findings suggest that serotonin exerts an inhibitory role in the control of nociception through activation of 5‐HT7 receptors, and point to a new potential therapeutic use of 5‐HT7 receptor agonists in the field of analgesia.

Pharmacological properties of S1RA, a new sigma-1 receptor antagonist that inhibits neuropathic pain and activity-induced spinal sensitization

BACKGROUND AND PURPOSE The sigma‐1 (σ1) receptor is a ligand‐regulated molecular chaperone that has been involved in pain, but there is limited understanding of the actions associated with its pharmacological modulation. Indeed, the selectivity and pharmacological properties of σ1 receptor ligands used as pharmacological tools are unclear and the demonstration that σ1 receptor antagonists have efficacy in reversing central sensitization‐related pain sensitivity is still missing.

Tetrodotoxin inhibits the development and expression of neuropathic pain induced by paclitaxel in mice

&NA; We evaluated the effect of low doses of systemically administered tetrodotoxin (TTX) on the development and expression of neuropathic pain induced by paclitaxel in mice. Treatment with paclitaxel (2 mg/kg, i.p., once daily during 5 days) produced long‐lasting (2–4 weeks) heat hyperalgesia (plantar test), mechanical allodynia (electronic Von Frey test) and cold allodynia (acetone drop method), with maximum effects observed on days 7, 10 and 10–14, respectively. Acute subcutaneous treatment with 1 or 3 μg/kg of TTX reduced the expression of mechanical allodynia, whereas higher doses (3 or 6 μg/kg) were required to reduce the expression of cold allodynia and heat hyperalgesia. In contrast, TTX (3 or 6 μg/kg, s.c.) did not affect the response to the same thermal and mechanical stimuli in control animals, which indicates that the antihyperalgesic and antiallodynic effects of TTX were not due to unspecific inhibition of the perception of these stimuli. Administration of TTX (6 μg/kg, s.c.) 30 min before each of the 5 doses of paclitaxel did not modify the development of heat hyperalgesia produced by the antineoplastic, but abolished the development of mechanical and cold allodynia. Coadministration of a lower dose of TTX (3 μg/kg) also prevented the development of mechanical allodynia. No signs of TTX‐induced toxicity or motor incoordination were observed. These data suggest that low doses of TTX can be useful to prevent and treat paclitaxel‐induced neuropathic pain, and that TTX‐sensitive subtypes of sodium channels play a role in the pathogenesis of chemotherapy‐induced neuropathic pain.

Selective Sigma-1 (σ1) Receptor Antagonists: Emerging Target for the Treatment of Neuropathic Pain

A large number of therapeutic roles have been proposed for sigma(1) receptors but the involvement of sigma(1) receptor in non-acute pain had not been well explored up to now. sigma(1) receptor knock-out mice became available offering us the possibility to study the role of sigma(1) receptor in nociception, particularly in models where central sensitization processes play a significant role. Given the attractive therapeutic potential, we have developed a chemical program aimed at the discovery of novel and selective sigma(1) ligands. Herein we discuss the rational basis of this approach and report preliminary pharmacological results of several chemical series and aspects of their structure-activity relationship on sigma(1) receptor. Functional data in pain models are presented mainly on one series that provide evidence to consider selective sigma(1) receptor antagonists an innovative and alternative approach for treating neuropathic pain.