Elisa Borsani

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.

Keratinocyte expression of calcitonin gene-related peptide β: implications for neuropathic and inflammatory pain mechanisms.

Summary In a variety of chronic pain conditions, calcitonin gene‐related peptide (CGRP) is expressed at high levels in the skin, blood, and cerebrospinal fluid. Our results reveal that much of the increase may be due to the CGRPβ isoform made by epidermal keratinocytes, which express high levels of CGRP immunoreactivity in a variety of human and animal chronic pain conditions. ABSTRACT Calcitonin gene‐related peptide (CGRP) is a vasodilatory peptide that has been detected at high levels in the skin, blood, and cerebrospinal fluid (CSF) under a variety of inflammatory and chronic pain conditions, presumably derived from peptidergic C and Aδ innervation. Herein, CGRP immunolabeling (IL) was detected in epidermal keratinocytes at levels that were especially high and widespread in the skin of humans from locations afflicted with postherpetic neuralgia (PHN) and complex region pain syndrome type 1 (CRPS), of monkeys infected with simian immunodeficiency virus, and of rats subjected to L5/L6 spinal nerve ligation, sciatic nerve chronic constriction, and subcutaneous injection of complete Freund’s adjuvant. Increased CGRP‐IL was also detected in epidermal keratinocytes of transgenic mice with keratin‐14 promoter driven overexpression of noggin, an antagonist to BMP‐4 signaling. Transcriptome microarray, quantitative Polymerase Chain Reaction (qPCR), and Western blot analyses using laser‐captured mouse epidermis from transgenics, monolayer cultures of human and mouse keratinocytes, and multilayer human keratinocyte organotypic cultures, revealed that keratinocytes express predominantly the beta isoform of CGRP. Cutaneous peptidergic innervation has been shown to express predominantly the alpha isoform of CGRP. Keratinocytes also express the cognate CGRP receptor components, Calcitonin receptor‐like receptor (CRLR), Receptor activity‐modifying protein 1 (RAMP1), CGRP‐receptor component protein (RCP) consistent with known observations that CGRP promotes several functional changes in keratinocytes, including proliferation and cytokine production. Our results indicate that keratinocyte‐derived CGRPβ may modulate epidermal homeostasis through autocrine/paracrine signaling and may contribute to chronic pain under pathological conditions.

Altered structure of small cerebral arteries in patients with essential hypertension.

Objective Structural alterations in the microcirculation may be considered an important mechanism of organ damage. An increased media-to-lumen ratio of subcutaneous small resistance arteries has been demonstrated to predict the development of cardiocerebrovascular events in hypertensive patients. Alterations in the structure of small cerebral arteries have been demonstrated in animal models of experimental or genetic hypertension. However, no evaluation with reliable techniques has ever been performed in humans. Design and methods Twenty-eight participants were included in the present study: they were 13 hypertensive patients and 15 normotensive individuals. All participants underwent a neurosurgical intervention for benign or malign tumors. A small portion of morphologically normal cerebral tissue was excised from surgical samples and examined. Cerebral small resistance arteries (relaxed diameter around 200 μm) were dissected and mounted on an isometric and isobaric myograph, and the tunica media to internal lumen ratio was measured. In addition, cerebral cortical microvessel density (MVD) was also evaluated. The tissue was sectioned and stained for CD31, and MVD was measured with an automated image analyzer (percentage of area stained). Blood pressure values were evaluated, before surgical intervention, by standard sphygmomanometry. Results M/L was significantly greater and MVD significantly lower in hypertensive patients than that in normotensive individuals. No difference between groups in collagen content or mechanical properties of cerebral small arteries was observed. Conclusion Our results indicate that structural alterations of small cerebral vessels are present in hypertensive patients compared with normotensive individuals, similar to those previously observed in subcutaneous small arteries.

Dose-dependent Mercuric Chloride Tubular Injury in Rat Kidney

Mercuric chloride (HgCl 2 ) produces an acute renal failure in experimental animal models. Since the mechanism of tubular injury has not completely been clarified, this morpho-quantitative study was undertaken to better understand the effects of 2 sublethal doses (T1=1 mg/kg and T3.5=3.5 mg/kg) of HgCl 2 in rat proximal tubules. Morphometrical analysis was performed to quantify both cytoplasmic and nuclear changes found in treated in respect to saline-injected proximal tubules. In the controls, single-cell damage was occasional and nucleolar changes were absent. HgCl 2 induced progressively severe proximal tubule atrophy. In the T1 group, necrosis was limited to pars recta cells and nucleolar segregation was often partial. In the T3.5 group, atrophy was extensive in both convoluted and straight tracts, the nucleolus was completely segregated and coiled body-like inclusions were detached from it. Ultrastructural analysis confirmed dose-dependent changes within straight proximal tubules, i.e., necrosis, apoptosis, nucleolar segregation, swollen mitochondria, vacuolization, and disrupted brush border. In conclusion, in the rat kidney HgCl 2 induced dose-dependent alterations not only in the cytoplasm but also in the nucleus of proximal tubule cells. These findings will be useful for better understanding of the pathogenesis of mercury nephrotoxicity and its genotoxic effect.

Intravenous neural stem cells abolish nociceptive hypersensitivity and trigger nerve regeneration in experimental neuropathy

Summary In experimental neuropathic pain, intravenous neural stem cells induce a rapid remission of hyperalgesia and allodynia, followed by improvement in nerve morphology. Abstract A nonphysiological repair of the lesioned nerve leading to the formation of neurinomas, altered nerve conduction, and spontaneous firing is considered the main cause of the events underlying neuropathic pain. It was investigated whether neural stem cell (NSCs) administration could lead to a physiological nerve repair, thus to a reduction of neuropathic pain symptoms such as hyperalgesia and allodynia in a well‐established model of this pain (sciatic nerve chronic constriction injury [CCI]). Moreover, since we and others showed that the peripheral nerve lesion starts a cascade of neuroinflammation‐related events that may maintain and worsen the original lesion, the effect of NSCs on sciatic nerve pro‐ and antiinflammatory cytokines in CCI mice was investigated. NSCs injected intravenously, when the pathology was already established, induced a significant reduction in allodynia and hyperalgesia already 3 days after administration, demonstrating a therapeutic effect that lasted for at least 28 days. Responses changed with the number of administered NSCs, and the effect on hyperalgesia could be boosted by a new NSC administration. Treatment significantly decreased proinflammatory, activated antiinflammatory cytokines in the sciatic nerve, and reduced spinal cord Fos expression in laminae I‐VI. Moreover, in NSC‐treated animals, a reparative process and an improvement of nerve morphology is present at a later time. Since NSC effect on pain symptoms preceded nerve repair and was maintained after cells had disappeared from the lesion site, we suggest that regenerative, behavioral, and immune NSC effects are largely due to microenvironmental changes they might induce at the lesion site.

Tubular Stress Proteins and Nitric Oxide Synthase Expression in Rat Kidney Exposed to Mercuric Chloride and Melatonin

Stress proteins such as HSP70 members (HSP72 and GRP75) and metallothionein (MT) protect the kidney against oxidative damage and harmful metals, whereas inducible nitric oxide synthase (iNOS) regulates tubular functions. A single dose of mercuric chloride (HgCl2) can cause acute renal failure in rats, its main target being the proximal tubule. Oxidative damage has been proposed as one of its pathogenic mechanisms. In this study we tested whether melatonin (MEL), a powerful antioxidant compound, is effective against HgCl2 nephrotoxicity. Rats were treated with saline, HgCl2 (3.5 mg/kg), MEL (5 mg/kg), and MEL + HgCl2 and examined after 24 hr for HSP72, GRP75, MT, and iNOS by immunohistochemistry and immunoblotting. Tubular effects of the treatment were then characterized by ultrastructure. In the HgCl2 group, all markers were overexpressed in convoluted proximal tubules and sometimes in distal tubules. In the MEL + HgCl2 group, GRP75 and iNOS decreased in convoluted and straight proximal tubules, whereas HSP72 and MT persisted more than the saline and MEL-only groups. Tubular damage and mitochondrial morphometry were improved by MEL pretreatment. In conclusion, the beneficial effect of MEL against HgCl2 nephrotoxicity was outlined morphologically and by the reduction of the tubular expression of stress proteins and iNOS. These markers could represent sensitive recovery index against mercury damage. (J Histochem Cytochem 54:1149-1157, 2006)

mGlu5 receptor antagonist decreases Fos expression in spinal neurons after noxious visceral stimulation

In this study we examined the effects of the glutamate metabotropic subtype 5 (mGlu5) receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) on Fos expression in the spinal cord in a model of visceral pain in the rat. We show that noxious stimulation increases the number of Fos-positive neurons in the dorsal horn of the thoracic and lumbar spinal cord, and that pretreatment with MPEP significantly reduces the number of Fos-positive neurons in these areas. These data indicate that mGlu5 is involved in the transmission of visceral pain in the spinal cord.

Controlling the activation of the Bv8/prokineticin system reduces neuroinflammation and abolishes thermal and tactile hyperalgesia in neuropathic animals

Chemokines are involved in neuroinflammation and contribute to chronic pain processing. The new chemokine prokineticin 2 (PROK2) and its receptors (PKR1 and PKR2) have a role in inflammatory pain and immunomodulation. In the present study, we investigated the involvement of PROK2 and its receptors in neuropathic pain.

Role of Carbon Monoxide and Biliverdin in Renal Ischemia/Reperfusion Injury

Heme oxygenase (HO) isoforms catalyze the conversion of heme to carbon monoxide (CO) and biliverdin/bilirubin with a concurrent release of iron. There is strong evidence that HO activity and products play a major role in renoprotection, however the exact molecular mechanisms underlying the beneficial effects exerted by this pathway are not fully understood. This review is aimed at illustrating the possible mechanism/s by which HO is renoprotective in the context of ischemia/reperfusion. We will first analyze the effects of exogenous administration of bilirubin/biliverdin and CO and then describe their biological activities once generated endogenously following stimulation of the HO pathway by either pharmacological means or gene targeting-mediated approaches.

Alterations of AQP2 expression in trigeminal ganglia in a murine inflammation model.

Aquaporins (AQPs) are small membrane channel proteins involved in osmoregulation. To date, only AQP1, AQP2, AQP4 and AQP9 have been found in the nervous system. Generally, they are involved in water movement in nervous tissue, nevertheless, recent data would suggest the involvement of AQPs in neurotransmission. In this work, we have evaluated the expression of AQP1 and AQP2 in the trigeminal ganglia of mice in an animal model of perioral acute inflammatory pain using immunohistochemistry and immunoblotting analysis. Our data have shown for the first time, the alteration of AQP2 expression in trigeminal ganglia in acute inflammatory pain showing increased and intracellular redistribution of AQP2 mainly in small-sized neurons and Schwann cells. Apart from this, the AQP1 expression remained unaltered. On the whole, these data support the hypothesis that AQP2 is involved in pain transmission in the peripheral nervous system.