Silvia Franchi

The soy isoflavone genistein reverses oxidative and inflammatory state, neuropathic pain, neurotrophic and vasculature deficits in diabetes mouse model.

Treatment of diabetes complications remains a substantial challenge. The aim of this study was to explore the ability of the soy isoflavone genistein in attenuating the signs that follow diabetes onset: nociceptive hypersensitivity, oxidative and inflammatory state, nerve growth factor (NGF) decrease and vascular dysfunctions. Genistein (3 and 6 mg/kg) was administered to C57BL/6J streptozotocin diabetic mice from the 2nd till the 5th week after disease induction. The hind paw withdrawal threshold to mechanical stimulation (tactile allodynia) was evaluated by a von Frey filament. The oxidative stress was assessed measuring: reactive oxygen species by fluorimetric analysis, both the lipoperoxide content, as malondialdehyde, the antioxidant enzymatic activities spectrophotometrically and the glutathione content spectrofluorimetrically. Proinflammatory cytokines and NGF were measured in the sciatic nerve by enzyme-linked immunosorbent assay. Aortic inducible (iNOS) and endothelial nitric oxide synthase (eNOS) protein content was measured by western immunoblotting. Genistein relieved diabetic peripheral painful neuropathy, reverted the proinflammatory cytokine and reactive oxygen species overproduction, and restored the NGF content in diabetic sciatic nerve. Furthermore it restored the GSH content and the GSH and GSSG ratio, improved the antioxidant enzymes activities, decreased reactive oxygen species and lipoperoxide level in the brain and liver. Finally it restored the iNOS and eNOS content and the superoxide dismutase activity in thoracic aorta. Hyperglycaemia and weight decrease were not affected. Genistein is able to reverse a diabetes established condition of allodynia, oxidative stress and inflammation, ameliorates NGF content and the vascular dysfunction, thus suggesting its possible therapeutic use for diabetes complications.

The purinergic antagonist PPADS reduces pain related behaviours and interleukin-1β, interleukin-6, iNOS and nNOS overproduction in central and peripheral nervous system after peripheral neuropathy in mice

&NA; Neuropathic pain consequent to peripheral injury is associated with local inflammation and overexpression of nitric oxide synthases (NOS) and inflammatory cytokines in locally recruited macrophages, Schwann and glial cells. We investigated the time course and localization of nitric oxide synthases (NOS) and cytokines in the central (spinal cord and thalamus) and peripheral nervous system (nerve and dorsal root ganglia), in a mouse model of mononeuropathy induced by sciatic nerve chronic constriction injury. ATP is recognized as an endogenous pain mediator. Therefore we also evaluated the role of purinergic signalling in pain hypersensitivity employing the P2 receptor antagonist, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), on pain behaviour, NOS and cytokines. The PPADS daily administration starting on day 3 after injury dose‐ and time‐dependently decreased both tactile allodynia and thermal hyperalgesia. PPADS (25 mg/kg) completely reversed nociceptive hypersensitivity and simultaneously reduced the increased NO/NOS system and IL‐1β in both peripheral (injured sciatic nerve and L4–L6 ipsilateral dorsal root ganglia) and central steps of nervous system (L4–L6 spinal cord and thalamus) involved in pain signalling. IL‐6 was overexpressed only in the peripheral nervous system and PPADS prolonged administration reduced it in sciatic nerve. In conclusion, we hypothesize that NO/NOS and IL‐1β have a pronociceptive role in this neuropathy model, and that purinergic antagonism reduces pain hypersensitivity by inhibiting their overactivity.

Buprenorphine ameliorates the effect of surgery on hypothalamus-pituitary-adrenal axis, natural killer cell activity and metastatic colonization in rats in comparison with morphine or fentanyl treatment.

Not all opioids employed in clinical practice share the same immunosuppressive properties. The potent partial micro-agonist buprenorphine appears to exhibit a neutral effect on the immune responses. Surgery stress is associated with decreased natural killer cell activity (NK) and enhancement of tumor metastasis in rats. We analyzed the ability of buprenorphine to prevent the effects of experimental surgery on HPA activation (plasma corticosterone levels), NK activity and lung diffusion of the NK sensitive tumor MADB106. Buprenorphine (0.1mg/kg) was compared with equianalgesic doses of fentanyl (0.1mg/kg) and morphine (10mg/kg) in this animal model. In normal animals morphine and fentanyl stimulate the HPA axis, decrease NK activity and augment tumor metastasis, while buprenorphine is devoid of these effects. Surgery significantly raised corticosterone levels, suppressed NK activity and increased MADB106 metastasis. Only buprenorphine was able to prevent the neuroendocrine and immune system alterations and ameliorate the increase of tumor metastasis induced by surgical stress. These preclinical findings suggest that an adequate treatment of surgically induced stress immunosuppression with an opioid drug devoid of immunosuppressive effects may also play a protective role against the metastatic diffusion following cancer surgery.

Bv8, the amphibian homologue of the mammalian prokineticins, induces a proinflammatory phenotype of mouse macrophages

The small protein Bv8, isolated from the amphibian skin, belongs to a novel family of secreted proteins linked to several biological effects. We describe the expression of Bv8/prokineticins and their receptors in mouse macrophages, and characterize their proinflammatory activities. The rodent analogue of Bv8, prokineticin‐2, is expressed by macrophages, as well as its G‐protein‐coupled receptor prokineticin receptor (PKR‐1 and PKR‐2). PKR‐1 is expressed more abundantly. Bv8 induces potent chemotaxis of macrophages at concentrations as low as 10−12 M. It stimulates lipopolysaccharide‐induced production of the proinflammatory cytokines IL‐1 and IL‐12, reducing that of the anti‐inflammatory cytokine IL‐10. The effects are observed starting at the very low concentration of 10−11 M. Effects on chemotaxis and cytokine are not pertussis‐toxin sensitive, but are completely prevented by addition of the phospholipase inhibitor U73122, suggesting a Gq protein is involved in the Bv8‐induced effects. Studies in PKR‐1 knockout mice indicate that all the activities exerted by Bv8 on macrophages are mediated by the PKR‐1 receptor. In conclusion, Bv8 appears to be able to induce the macrophage to migrate and to acquire a proinflammatory phenotype.

Genistein, a natural phytoestrogen from soy, relieves neuropathic pain following chronic constriction sciatic nerve injury in mice : anti-inflammatory and antioxidant activity

There is great interest in soy isoflavones as alternatives to endogenous estrogens not only in hormonal pathologies, but also in inflammatory, neurodegenerative diseases, and pain. We investigated the effect of the isoflavone genistein on neuropathic pain. Genistein binds estrogen receptors (ER) with higher affinity for the ERβ particularly expressed in neuronal and immune cells. Neuropathy was induced in mice by means of chronic sciatic nerve constriction, and the subcutaneous administration of genistein from the third day after the lesion reversed pain hypersensitivity in a time‐ and dose‐dependent manner. This effect may have been due to the activation of classical nuclear receptor and/or anti‐oxidant, anti‐inflammatory, and immunomodulating properties of genistein. The fact that a specific ERβ antagonist prevented both its anti‐allodynic and anti‐hyperalgesic action, whereas a specific ERα antagonist was ineffective and a non‐selective ER antagonist only reversed the anti‐allodynic effect, suggests the involvement of ERβ. Antioxidant effects are also involved as the anti‐nociceptive dose reversed the increase in reactive oxygen species and malondialdehyde in injured paw tissues, and increased the activity of anti‐oxidant enzymes. The phytoestrogen had immunomodulatory and anti‐inflammatory activities as it reduced peripheral and central nuclear factor‐κB, nitric oxide system and pro‐inflammatory cytokine over‐activation. Taken together, our results suggest that genistein could ameliorate painful neuropathy by multiple mechanisms.

Acute and late changes in intraarticular cytokine levels following anterior cruciate ligament injury.

Surgical reconstruction of the anterior cruciate ligament (ACL) does not necessarily decrease the risk of developing osteoarthritis (OA). The inflammatory response and relative changes in pro‐ and anti‐inflammatory cytokines could participate in triggering the development of OA. To test this hypothesis we measured the concentrations of IL‐1β, IL‐1ra, IL‐6, IL‐8, IL‐10, and TNF‐α at different times after ACL rupture. The sample population consisted of 48 patients with ACL tear which were assigned to different groups according to the time elapsed from the injury: 22 acute (A), 7 early sub‐acute (ESA), 11 late sub‐acute (LSA), and 8 chronic (C). In group A, there were high levels of IL‐1β, IL‐6, and IL‐8, whereas levels of IL‐1ra and TNF‐α were significantly lower than usually reported. IL‐1β and IL‐8 concentrations returned with time to normal levels in the ESA group. Interestingly, IL‐1ra levels remained always significantly lower than normally reported levels, and TNF‐α levels did not increase after trauma. Our data show increased level of pro‐inflammatory cytokines (IL‐6 and IL‐8) in the acute phase of inflammation which could be responsible for triggering cartilage catabolism and suggest that prompt neutralization of IL‐6 and IL‐8 accumulations in synovial fluid could help prevent development of OA in ACL‐injured knees.

Transient early expression of TNF-α in sciatic nerve and dorsal root ganglia in a mouse model of painful peripheral neuropathy

The proinflammatory cytokine tumor necrosis factor-alpha (TNF) is an important mediator in neuropathic pain. We investigated the temporal pattern of TNF mRNA expression in the sciatic nerve, in dorsal root ganglia (DRG) and spinal cord in the mouse chronic constriction injury model of neuropathy with quantitative real-time polymerase chain reaction. Neuropathic pain-like behaviour was monitored by evaluating thermal hyperalgesia and mechanical allodynia. Pain-related behaviour and TNF expression were evaluated 6 h, 1, 3, 7 and 14 days after injury. Naive animals and sham-operated mice were used as controls. We found an early upregulation of sciatic nerve TNF mRNA levels in chronic constriction injury (CCI) and sham-operated animals 6 h after surgery: 1 day later TNF overexpression was present in CCI mice only and disappeared 3 days after injury. The mRNA cytokine levels were elevated in DRG 1 and 3 days after surgery in CCI animals only, while the cytokine was not modulated in the spinal cord. A significant hyperalgesia was present in CCI and sham-operated mice at 6 h and 1 day, while at later time point only CCI mice presented lower thresholds. Mechanical allodynia was already present only in CCI animals 6 h from surgery and remained constant up to the 14 th day. The results indicate that a transient early TNF upregulation takes place in peripheral nervous system after CCI that can activate a cascade of proinflammatory/pronociceptive mediators.

A pilot trial of low-dose naltrexone in primary progressive multiple sclerosis:

A sixth month phase II multicenter-pilot trial with a low dose of the opiate antagonist Naltrexone (LDN) has been carried out in 40 patients with primary progressive multiple sclerosis (PPMS). The primary end points were safety and tolerability. Secondary outcomes were efficacy on spasticity, pain, fatigue, depression, and quality of life. Clinical and biochemical evaluations were serially performed. Protein concentration of β-endorphins (BE) and mRNA levels and allelic variants of the μ-opiod receptor gene (OPRM1) were analyzed. Five dropouts and two major adverse events occurred. The remaining adverse events did not interfere with daily living. Neurological disability progressed in only one patient. A significant reduction of spasticity was measured at the end of the trial. BE concentration increased during the trial, but no association was found between OPRM1 variants and improvement of spasticity. Our data clearly indicate that LDN is safe and well tolerated in patients with PPMS.

Cytokine Modulation is Necessary for Efficacious Treatment of Experimental Neuropathic Pain

Neuropathic pain originates from a damage or disease affecting the somatosensory system. Its treatment is unsatisfactory as it appears refractory to most analgesics. Animal models of neuropathic pain are now available that help to clarify the underlying mechanisms. Recently it has been recognized that inflammatory and immune mechanisms in the peripheral and in the central nervous system play a role in the onset and the maintenance of pain. In response to nervous tissue damage, activation of resident or recruited immune cells leads to the production of inflammatory mediators, as cytokines. In models of neuropathic pain, such as nerve injury and diabetes induced neuropathy, the time course of the expression of the proinflammatory cytokines TNF-α,IL-1β and IL-6 and of the antiinflammatory cytokine IL-10 has been well characterized both in the peripheral (sciatic nerve, dorsal root ganglia) and the central (spinal cord) nervous system. These cytokines appear activated/modulated in the nervous tissue in parallel with the occurrence of painful behaviour, i.e. allodynia and hyperalgesia. Novel therapeutic approaches efficacious to reduce painful symptoms, for example treatments with the non specific purinergic antagonist PPADS, the phytoestrogen genistein and a cell stem therapy with murine adult neural stem cells also re-established a balance between pro and antinflammatory mediators in the peripheral and central nervous system. These data suggest a pivotal role of immune system and inflammation in neuropathic pain. The modulation of inflammatory molecules appears to be a common trait accomplished throughout different mechanisms by different drugs that might converge in neuropathic pain modulation.

Increased tumor necrosis factor-alpha and prostaglandin E2 concentrations in the cerebrospinal fluid of rats with inflammatory hyperalgesia: the effects of analgesic drugs.

BACKGROUND:We examined the changes in cerebrospinal fluid (CSF) concentrations of prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α) after intraplantar administration of complete Freund’s adjuvant (CFA) in rats. In addition, we investigated whether different analgesic drugs orally administered at antihyperalgesic doses were able to prevent the changes in PGE2 and TNF-α spinal levels associated with hindpaw inflammation. METHODS:The Randall–Selitto paw-withdrawal test was used to measure inflammatory hyperalgesia. Tramadol (7.5 mg/kg), paracetamol (65 mg/kg), tramadol plus paracetamol and nimesulide (5 mg/kg) were administered orally twice a day, starting from the first day after the CFA injection. PGE2 in the CSF was measured by enzyme immunoassay, and TNF-α by ELISA. Behavioral and biochemical parameters were measured on Day 7 after intraplantar injection of CFA or saline. RESULTS:Withdrawal thresholds to mechanical stimuli decreased markedly in the CFA-treated paw. In these animals the quantification of proinflammatory mediators in the CSF revealed a significant increase in both PGE2 and TNF-α concentrations. All the pharmacological treatments prevented the development of the hyperalgesia as well as the PGE2 increase in the CSF. Conversely, a prevention of the increase in TNF-α levels was observed only in rats treated with nimesulide or tramadol and paracetamol in combination. CONCLUSIONS:Our results demonstrate that peripheral inflammatory hyperalgesia is associated with significant changes of proinflammatory mediators in the CSF. It is important to note, however, that spinal PGE2 and TNF-α proved to be differently affected by pharmacological treatments able to fully abolish the hyperalgesia.