Endothelial-to-Mesenchymal Transition Contributes to Accelerated Atherosclerosis in Hutchinson-Gilford Progeria Syndrome.

BACKGROUND: Atherosclerosis is the main medical problem in Hutchinson-Gilford progeria syndrome, a rare premature aging disorder caused by the mutant lamin-A protein progerin. Recently, we found that limiting progerin expression to vascular smooth muscle cells (VSMCs) is sufficient to hasten atherosclerosis and death in Apoe-deficient mice. However, the impact of progerin-driven VSMC defects on endothelial cells (ECs) remained unclear. METHODS: Apoe- or Ldlr-deficient C57BL/6J mice with ubiquitous, VSMC-, EC- or myeloid-specific progerin expression fed a normal or high-fat diet were used to study endothelial phenotype during Hutchinson-Gilford progeria syndrome-associated atherosclerosis. Endothelial permeability to low-density lipoproteins was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein and confocal microscopy analysis of the aorta. Leukocyte recruitment to the aortic wall was evaluated by en face immunofluorescence. Endothelial-to-mesenchymal transition (EndMT) was assessed by quantitative polymerase chain reaction and RNA sequencing in the aortic intima and by immunofluorescence in aortic root sections. TGFß (transforming growth factor ß) signaling was analyzed by multiplex immunoassay in serum, by Western blot in the aorta, and by immunofluorescence in aortic root sections. The therapeutic benefit of TGFß1/SMAD3 pathway inhibition was evaluated in mice by intraperitoneal injection of SIS3 (specific inhibitor of SMAD3), and vascular phenotype was assessed by Oil Red O staining, histology, and immunofluorescence in the aorta and the aortic root. RESULTS: Both ubiquitous and VSMC-specific progerin expression in Apoe-null mice provoked alterations in aortic ECs, including increased permeability to low-density lipoprotein and leukocyte recruitment. Atherosclerotic lesions in these progeroid mouse models, but not in EC- and myeloid-specific progeria models, contained abundant cells combining endothelial and mesenchymal features, indicating extensive EndMT triggered by dysfunctional VSMCs. Accordingly, the intima of ubiquitous and VSMC-specific progeroid models at the onset of atherosclerosis presented increased expression of EndMT-linked genes, especially those specific to fibroblasts and extracellular matrix. Aorta in both models showed activation of the TGFß1/SMAD3 pathway, a major trigger of EndMT, and treatment of VSMC-specific progeroid mice with SIS3 alleviated the aortic phenotype. CONCLUSIONS: Progerin-induced VSMC alterations promote EC dysfunction and EndMT through TGFß1/SMAD3, identifying this process as a candidate target for Hutchinson-Gilford progeria syndrome treatment. These findings also provide insight into the complex role of EndMT during atherogenesis.

Cholesterol lowering depletes atherosclerotic lesions of smooth muscle cell-derived fibromyocytes and chondromyocytes.

Drugs that lower plasma apolipoprotein B (ApoB)-containing lipoproteins are central to treating advanced atherosclerosis and provide partial protection against clinical events. Previous research showed that lowering ApoB-containing lipoproteins stops plaque inflammation, but how these drugs affect the heterogeneous population of plaque cells derived from smooth muscle cells (SMCs) is unknown. SMC-derived cells are the main cellular component of atherosclerotic lesions and the source of structural components that determine the size of plaques and their propensity to rupture and trigger thrombosis, the proximate cause of heart attack and stroke. Using lineage tracing and single-cell techniques to investigate the full SMC-derived cellular compartment in progressing and regressing plaques in mice, here we show that lowering ApoB-containing lipoproteins reduces nuclear factor kappa-light-chain-enhancer of activated B cells signaling in SMC-derived fibromyocytes and chondromyocytes and leads to depletion of these abundant cell types from plaques. These results uncover an important mechanism through which cholesterol-lowering drugs can achieve plaque regression.

Solvothermal Synthesis of Cu2ZnSnSe4 Nanoparticles and Their Visible-Light-Driven Photocatalytic Activity.

Cu2ZnSnSe4 (CZTSe) nanoparticles (NPs) were successfully synthesized via a solvothermal method. Their structural, compositional, morphological, optoelectronic, and electrochemical properties have been characterized by X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), Field-emission scanning electron microscopy (FE-SEM), transmission electron microscope (TEM), UV-vis absorption spectroscopy, and electrochemical impedance spectroscopy (EIS) techniques. Porosimetry and specific surface area in terms of the Brunauer-Emmett-Teller (BET) technique have also been studied. XRD indicates the formation of a polycrystalline kesterite CZTSe phase. Raman peaks at 173 and 190 cm-1 confirm the formation of a pure phase. TEM micrographs revealed the presence of nanoparticles with average sizes of ~90 nm. A BET surface area of 7 m2/g was determined. The CZTSe NPs showed a bandgap of 1.0 eV and a p-type semiconducting behavior. As a proof of concept, for the first time, the CZTSe NPs have been used as a visible-light-driven photocatalyst to Congo red (CR) azo dye degradation. The nanophotocatalyst material under simulated sunlight results in almost complete degradation (96%) of CR dye after 70 min, following a pseudo-second-order kinetic model (rate constant of 0.334 min-1). The prepared CZTSe was reusable and can be repeatedly used to remove CR dye from aqueous solutions.

Sphingomyelinase-responsive nanomicelles for targeting atherosclerosis.

Atherosclerosis, a leading cause of cardiovascular diseases requires approaches to enhance disease monitoring and treatment. Nanoparticles offer promising potential in this area by being customisable to target components or molecular processes within plaques, while carrying diagnostic and therapeutic agents. However, the number of biomarkers available to target this disease is limited. This study investigated the use of sphingomyelin-based nanomicelles triggered by sphingomyelinase (SMase) in atherosclerotic plaques. Accumulation of iron oxide-based nanomicelles in the plaque was demonstrated by fluorescence, MR imaging and electron microscopy. These findings demonstrate the possibility of utilising SMase as a mechanism to retain nanoprobes within plaques, thus opening up possibilities for future therapeutic interventions.

One-Step Hydrothermal Synthesis of Cu2ZnSnS4 Nanoparticles as an Efficient Visible Light Photocatalyst for the Degradation of Congo Red Azo Dye.

A hydrothermal method was successfully employed to synthesize kesterite Cu2ZnSnS4 (CZTS) nanoparticles. X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), field-emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), and optical ultraviolet-visible (UV-vis) spectroscopy were used for characterization of structural, chemical, morphological, and optical properties. XRD results confirmed that a nanocrystalline CZTS phase corresponding to the kesterite structure was formed. Raman analysis confirmed the existence of single pure phase CZTS. XPS results revealed the oxidation states as Cu+, Zn2+, Sn4+, and S2-. FESEM and TEM micrograph images revealed the presence of nanoparticles with average sizes between 7 nm to 60 nm. The synthesized CZTS nanoparticles bandgap was found to be 1.5 eV which is optimal for solar photocatalytic degradation applications. The properties as a semiconductor material were evaluated through the Mott-Schottky analysis. The photocatalytic activity of CZTS has been investigated through photodegradation of Congo red azo dye solution under solar simulation light irradiation, proving to be an excellent photo-catalyst for CR where 90.2% degradation could be achieved in just 60 min. Furthermore, the prepared CZTS was reusable and can be repeatedly used to remove Congo red dye from aqueous solutions.

New 3-Dimensional Volumetric Ultrasound Method for Accurate Quantification of Atherosclerotic Plaque Volume.

BACKGROUND: Carotid and femoral plaque burden is a recognized biomarker of cardiovascular disease risk. A new electronic-sweep 3-dimensional (3D)-matrix transducer method can improve the functionality and image quality of vascular ultrasound atherosclerosis imaging. OBJECTIVES: This study aimed to validate this method for plaque volume measurement in early and intermediate-advanced plaques in the carotid and femoral territories. METHODS: Plaque volumes were measured ex vivo in pig carotid and femoral artery specimens by 3-dimensional vascular ultrasound (3DVUS) using a 3D-matrix (electronic-sweep) transducer and its associated 3D plaque quantification software, and were compared with gold-standard histology. To test the clinical feasibility and accuracy of the 3D-matrix transducer, an experiment was conducted in intermediate-high risk individuals with carotid and femoral atherosclerosis. The results were compared with those obtained using the previously validated mechanical-sweep 3D transducer and established 2-dimensional (2D)-based plaque quantification software. RESULTS: In the ex vivo study, the authors assessed 19 atherosclerotic plaques (plaque volume, 0.76 µL-56.30 µL), finding strong agreement between measurements with the 3D-matrix transducer and the histological gold-standard (intraclass correlation coefficient [ICC]: 0.992; [95% CI: 0.978-0.997]). In the clinical analysis of 20 patients (mean age 74.6 ± 4.45 years; 40% men), the authors found 64 (36 carotid and 28 femoral) of 80 scanned territories with atherosclerosis (measured atherosclerotic volume, 10 µL-859 µL). There was strong agreement between measurements made from electronic-sweep and mechanical-sweep 3DVUS transducers (ICC: 0.997 [95% CI: 0.995-0.998]). Agreement was also high between plaque volumes estimated by the 2D and 3D plaque quantification software applications (ICC: 0.999 [95% CI: 0.998-0.999]). Analysis time was significantly shorter with the 3D plaque quantification software than with the 2D multislice approach with a mean time reduction of 46%. CONCLUSIONS: 3DVUS using new matrix transducer technology, together with improved 3D plaque quantification software, simplifies the accurate volume measurement of early (small) and intermediate-advanced plaques located in carotid and femoral arteries.

Analysis of 18F-Sodium Fluoride Positron Emission Tomography Signal Sources in Atherosclerotic Minipigs Shows Specific Binding of 18F-Sodium Fluoride to Plaque Calcifications.

Objective: 18F-sodium fluoride (18F-NaF) positron emission tomography (PET) imaging is thought to visualize active atherosclerotic plaque calcification. This is supported by the binding of 18F-NaF to plaque calcification ex vivo, but no prior studies have examined binding of 18F-NaF to human-like plaque in vivo. Our aim was to validate the specificity of 18F-NaF PET for plaque calcifications in atherosclerotic minipigs. Approach and Results: Gain-of-function PCSK9D374Y (proprotein convertase/subtilisin kexin type 9) transgenic Yucatan minipigs (n=4) were fed high-fat diet for 2.5 years to develop atherosclerosis and then subjected to 18F-NaF PET/computed tomography imaging. The heart, aorta, and iliac arteries were immediately re-scanned ex vivo after surgical extraction. Lesions from the abdominal aorta, iliac arteries, and coronary arteries were cryo-sectioned for autoradiography. Histological plaque characteristics, PET/computed tomography signal, and autoradiography were linked through regression and co-localization analysis. Arterial 18F-NaF PET signal had intensities comparable to clinical scans and colocalized moderately with calcification detected by computed tomography. Histological analysis showed calcification spanning from microcalcifications near lipid pools and necrotic core to more homogenous macrocalcifications. Comparison with arteries from autopsy cases confirmed the resemblance in localization and appearance with early human plaque calcification. Regression analysis in the abdominal aorta showed correlations with calcified plaque but could not rule out contributions from noncalcified plaque. This was resolved by autoradiography, which showed specific accumulation in plaque calcifications in all examined arteries. In the context of porcine abdominal aorta, 18F-NaF PET imaging was, however, less accurate than computed tomography for detecting small calcifications. Conclusions: 18F-NaF accumulates specifically in calcifications of atherosclerotic plaques in vivo.

Dianhydrogalactitol Overcomes Multiple Temozolomide Resistance Mechanisms in Glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive primary tumor type in the central nervous system in adults. Resistance to chemotherapy remains one of the major obstacles in GBM treatment. Identifying and overcoming the mechanisms of therapy resistance is instrumental to develop novel therapeutic approaches for patients with GBM. To determine the major drivers of temozolomide (TMZ) sensitivity, we performed shRNA screenings in GBM lines with different O6-methylguanine-DNA methyl-transferase (MGMT) status. We then evaluated dianhydrogalactitol (Val-083), a small alkylating molecule that induces interstrand DNA crosslinking, as a potential treatment to bypass TMZ-resistance mechanisms. We found that loss of mismatch repair (MMR) components and MGMT expression are mutually exclusive mechanisms driving TMZ resistance in vitro Treatment of established GBM cells and tumorsphere lines with Val-083 induces DNA damage and cell-cycle arrest in G2-M phase, independently of MGMT or MMR status, thus circumventing conventional resistance mechanisms to TMZ. Combination of TMZ and Val-083 shows a synergic cytotoxic effect in tumor cells in vitro, ex vivo, and in vivo We propose this combinatorial treatment as a potential approach for patients with GBM.

Effects of castration on atherosclerosis in Yucatan minipigs with genetic hypercholesterolemia.

BACKGROUND: Low plasma testosterone, either spontaneous or as a result of androgen deprivation therapy for prostate cancer, is associated with an increased risk of cardiovascular events. The underlying mechanism in humans is not understood. Experimental studies in mice have shown that castration facilitates atherogenesis and may increase signs of plaque vulnerability. Pigs used for translational atherosclerosis research have frequently been castrated for practical or commercial reasons, but the effect of castration on atherosclerosis has never been systematically evaluated in pigs. OBJECTIVE: To study the effect of castration on atherosclerotic plaque burden and type in genetically modified minipigs with hypercholesterolemia. METHODS: Newborn male Yucatan minipigs with transgenic overexpression of a human gain-of-function mutant of proprotein convertase subtilisin/kexin type 9 were randomized to undergo orchiectomy (n = 8) or serve as controls (n = 6). Minipigs were started on high-fat diet at 3 months of age and the amount and composition of atherosclerotic lesions were analyzed at 12 months of age. Plasma lipid profiles and behavioral parameters were also assessed. RESULTS: Plasma lipids were slightly affected to a more atherogenic profile by orchiectomy, but atherosclerotic lesion size was unaltered in the LAD, thoracic aorta, abdominal aorta, and iliac arteries. The distribution of lesion types (xanthomas, pathological intimal thickening and fibroatheromas) were also not statistically different between groups in any of the examined vascular territories. The abdominal aorta developed the most advanced stages of disease with reproducible fibroatheroma formation, and here it was found that the area of necrotic core was significantly increased in orchiectomized pigs compared with controls. Orchiectomy also reduced aggressive behavior. CONCLUSIONS: Castration does not alter the burden of atherosclerosis in hypercholesterolemic Yucatan minipigs, but may increase necrotic core area in fibroatheromas.

Vascular Smooth Muscle-Specific Progerin Expression Accelerates Atherosclerosis and Death in a Mouse Model of Hutchinson-Gilford Progeria Syndrome.

BACKGROUND: Progerin, an aberrant protein that accumulates with age, causes the rare genetic disease Hutchinson-Gilford progeria syndrome (HGPS). Patients who have HGPS exhibit ubiquitous progerin expression, accelerated aging and atherosclerosis, and die in their early teens, mainly of myocardial infarction or stroke. The mechanisms underlying progerin-induced atherosclerosis remain unexplored, in part, because of the lack of appropriate animal models. METHODS: We generated an atherosclerosis-prone model of HGPS by crossing apolipoprotein E-deficient (Apoe-/-) mice with LmnaG609G/G609G mice ubiquitously expressing progerin. To induce progerin expression specifically in macrophages or vascular smooth muscle cells (VSMCs), we crossed Apoe-/-LmnaLCS/LCS mice with LysMCre and SM22αCre mice, respectively. Progerin expression was evaluated by polymerase chain reaction and immunofluorescence. Cardiovascular alterations were determined by immunofluorescence and histology in male mice fed normal chow or a high-fat diet. In vivo low-density lipoprotein retention was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein. Cardiac electric defects were evaluated by electrocardiography. RESULTS: Apoe-/-LmnaG609G/G609G mice with ubiquitous progerin expression exhibited a premature aging phenotype that included failure to thrive and shortened survival. In addition, high-fat diet-fed Apoe-/-LmnaG609G/G609G mice developed a severe vascular pathology, including medial VSMC loss and lipid retention, adventitial fibrosis, and accelerated atherosclerosis, thus resembling most aspects of cardiovascular disease observed in patients with HGPS. The same vascular alterations were also observed in Apoe-/-LmnaLCS/LCSSM22αCre mice expressing progerin specifically in VSMCs, but not in Apoe-/-LmnaLCS/LCSLysMCre mice with macrophage-specific progerin expression. Moreover, Apoe-/-LmnaLCS/LCSSM22αCre mice had a shortened lifespan despite the lack of any overt aging phenotype. Aortas of ubiquitously and VSMC-specific progerin-expressing mice exhibited increased retention of fluorescently labeled human low-density lipoprotein, and atheromata in both models showed vulnerable plaque features. Immunohistopathological examination indicated that Apoe-/-LmnaLCS/LCSSM22αCre mice, unlike Apoe-/-LmnaG609G/G609G mice, die of atherosclerosis-related causes. CONCLUSIONS: We have generated the first mouse model of progerin-induced atherosclerosis acceleration, and demonstrate that restricting progerin expression to VSMCs is sufficient to accelerate atherosclerosis, trigger plaque vulnerability, and reduce lifespan. Our results identify progerin-induced VSMC death as a major factor triggering atherosclerosis and premature death in HGPS.

Delayed postoperative latent pain sensitization revealed by the systemic administration of opioid antagonists in mice.

The long-lasting post-surgical changes in nociceptive thresholds in mice, indicative of latent pain sensitization, were studied. The contribution of kappa opioid and N-methyl-d-aspartate (NMDA) receptors was assessed by the administration of nor-binaltorphimine or MK-801; dynorphin levels in the spinal cord were also determined. Animals underwent a plantar incision and/or a subcutaneous infusion of remifentanil (80µg/kg), and mechanical thresholds (von Frey) were evaluated at different times. On day 21, after complete recovery of mechanical thresholds and healing of the wound, one of the following drugs was administered subcutaneously: (-)-naloxone (1mg/kg), (+)-naloxone (1mg/kg), naloxone-methiodide (3mg/kg), or nor-binaltorphimine (5mg/kg). Another group received subcutaneous MK-801 (0.15mg/kg) before nor-binaltorphimine administration. Dynorphin on day 21 was determined in the spinal cord by immunoassay. In mice receiving remifentanil during surgery, the administration of (-)-naloxone or nor-binaltorphimine induced significant hyperalgesia even 5months after manipulation. Nociceptive thresholds remained unaltered after (+)-naloxone or naloxone-methiodide. On day 21 after manipulation, the administration of MK-801 prevented nor-binaltorphimine-induced hyperalgesia. No changes in dynorphin levels were observed before or after opioid antagonist administration. In conclusion, surgery produced latent pain sensitization evidenced by opioid antagonist-precipitated hyperalgesia. The effect was stereospecific, centrally originated, and mediated by kappa opioid receptors. The blockade of nor-binaltorphimine-induced hyperalgesia by MK-801, suggests that NMDA receptors are also involved. Our results show for the first time that surgery induces latent, long-lasting changes in the processing of nociceptive information that can be induced by non-nociceptive stimuli such as the administration of opioid antagonists.

Comparison of commercially-available RNA extraction methods for effective bacterial RNA isolation from milk spiked samples

Nucleic-acid based methods for bacterial identification are extremely useful in diagnostic applications due to their specificity and sensitivity. However, they require an optimal purification of the target molecules. As part of the development of a new diagnostic method for the detection of bacterial RNA in cow milk, we have compared four commercially available RNA extraction kits for the isolation of bacterial RNA from spiked UHT milk samples. The kits were compared in terms of extraction efficiency and RNA purity using two bacterial species, the Gram negative Escherichia coli and the Gram positive Staphylococcus aureus. Two kits are based in silica-matrix extraction, and the other two in the guanidinium thiocyanate-phenol-chloroform extraction. In our hands, the RNeasy Protect Bacteria Mini kit from QIAGEN was the best performing in terms of RNA yield, quality, reproducibility and consumable needs, under the conditions here described.

Increased spinal dynorphin levels and phospho-extracellular signal-regulated kinases 1 and 2 and c-Fos immunoreactivity after surgery under remifentanil anesthesia in mice.

In humans, remifentanil anesthesia enhances nociceptive sensitization in the postoperative period. We hypothesized that activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the expression of c-Fos, prodynorphin (mRNA), and dynorphin in the spinal cord could participate in the molecular mechanisms underlying postoperative opioid-induced sensitization. In a mouse model of incisional pain, we evaluated thermal (Hargreaves test) and mechanical (von Frey) hyperalgesia during the first 21 postoperative days. Moreover, prodynorphin (mRNA, real-time polymerase chain reaction), dynorphin (enzymatic immunoassay), c-Fos expression, and ERK1/2 phosphorylation (both by immunohistochemistry) in the lumbar spinal cord were assessed. Surgery performed under remifentanil anesthesia induced a maximal decrease in nociceptive thresholds between 4 h and 2 days postoperatively (p < 0.001) that lasted 10 to 14 days compared with noninjured animals. In the same experimental conditions, a significant increase in prodynorphin mRNA expression (at 2 and 4 days) followed by a sustained increase of dynorphin (days 2 to 10) in the spinal cord was observed. We also identified an early expression of c-Fos immunoreactivity in the superficial laminae of the dorsal horn of the spinal cord (peak at 4 h; p < 0.001), together with a partial activation of ERK1/2 (4 h; p < 0.001). These findings suggest that activated ERK1/2 could induce c-Fos expression and trigger the transcription of prodynorphin in the spinal cord. This in turn would result in long-lasting increased levels of dynorphin that, in our model, could participate in the persistence of pain but not in the manifestation of first pain.

Deletion of the inducible nitric oxide synthase gene reduces peripheral morphine tolerance in a mouse model of chronic inflammation.

The implication of inducible nitric-oxide synthase (iNOS) on peripheral tolerance to morphine was evaluated in wild-type (WT) and iNOS knockout mice. Chronic inflammation was induced by subplantar (s.p.) injection of Complete Freund's Adjuvant (CFA), and morphine tolerance by subcutaneous implantation of a 75 mg morphine-pellet. Withdrawal was assessed after the intraperitoneal injection of 2 mg/kg naloxone. Antinociception was assessed (Randall-Selitto test) 5 min after a fixed dose of s.p. morphine (16 microg). In the absence of inflammation, s.p. morphine did not induce antinociception, while during CFA-inflammation produced 47.4 +/- 0.8 and 38.8 +/- 2.7% inhibitions respectively, in each genotype (P < 0.05). In morphine-tolerant mice with CFA-inflammation, no antinociception could be elicited in WT mice (2.4 +/- 0.3% inhibition); however, iNOS knockout mice showed significant antinociception (33.1 +/- 0.9%) (P < 0.001). Thus, iNOS gene deletion partially prevented tolerance to the peripheral effects of morphine, and significantly attenuated withdrawal-induced hyperactivity.

Effects of chronic inflammation and morphine tolerance on the expression of phospho-ERK 1/2 and phospho-P38 in the injured tissue.

Opioids are used in humans in the treatment of chronic osteoarticular pain, but the development of tolerance to the analgesic effects after continuous administration is still not well understood. The aim of the present study was to evaluate the expression of phospho-ERK 1/2 and phospho-p38 in mice with monoarthritis chronically exposed to morphine as a possible explanation for the development of tolerance. Inflammation was induced by intraplantar injection of complete Freund's adjuvant (CFA) and the tolerance by implantation of 75 mg morphine pellets. The results of the present study show that ERKs phosphorylation is unaltered by inflammation or morphine tolerance, each one individually, in the plantar tissue. In contrast, phospho-p38 is similarly decreased by inflammation or morphine tolerance. In naïve but not in tolerant animals, acute injection of morphine induces significant increase in phospho-p38 without any changes in phospho-ERK 1/2 expression. During inflammation, the acute injection of morphine induces a significant increase in the expression of ERK 1/2, but not in phospho-p38, in naïve animals. Phospho-ERK 1/2 expression was significantly decreased in the presence of inflammation plus tolerance. In contrast, no significant differences in phospho-p38 expression were observed between naïve and tolerant animals acutely injected with saline or morphine in presence of CFA inflammation. These results suggest that ERK but not p38 could be implicated in the development of morphine tolerance during peripheral inflammation. These experiments could contribute to establish the mechanisms implicated in the development of morphine tolerance in presence of inflammatory pain.

Tolerance to the antinociceptive effects of peripherally administered opioids. Expression of beta-arrestins.

Tolerance to peripheral antinociception after chronic exposure to systemic morphine was assessed in mice with chronic CFA-inflammation; cross-tolerance to locally administered mu, delta and kappa-opioid agonists and levels of beta-arrestins in the injured paw, were also evaluated. Tolerance was induced by the subcutaneous implantation of a 75 mg morphine-pellet, and antinociception evaluated with the Randall-Selitto test, 5 min after the subplantar injection of morphine, fentanyl, buprenorphine, DPDPE, U-50488H or CRF. Experiments were performed in the absence and presence of CFA-inflammation, in animals implanted with a morphine or placebo pellet. Beta-arrestin protein levels were determined by western blot. In mice without inflammation, subplantar opioids did not induce antinociception, while during CFA-inflammation, all drugs generated dose-response curves with an order of potency of: U-50488H < DPDPE < morphine < buprenorphine < fentanyl << CRF. During CFA-inflammation plus morphine-pellet, the potency of fentanyl decreased 1.25 times, while that of DPDPE, U-50488H and CRF diminished approximately 2.5-4.3 times. For each drug, the ratio between the ED(50)'s in tolerant and naive animals, was significantly higher than 1 (except for buprenorphine and fentanyl), demonstrating partial cross-tolerance to systemic morphine. Inflammation induced a twofold increase in beta-arrestin expression (p<0.01), and the levels decreased after acute morphine exposure (p<0.05). Tolerance did not alter beta-arrestins, but partially prevented the increase induced by inflammation. The results suggest that peripheral beta-arrestins could facilitate peripheral OR-desensitization and tolerance development. Clinically, the experiments could be useful to establish the effectiveness of local opioid administration in patients with musculoskeletal pain, chronically receiving morphine analgesia.

The pro-nociceptive effects of remifentanil or surgical injury in mice are associated with a decrease in delta-opioid receptor mRNA levels: Prevention of the nociceptive response by on-site delivery of enkephalins.

The ultra-short-acting mu-opioid receptor (MOR) agonist remifentanil enhances postsurgical pain when used as main anesthetic in animal models and man. Although the mechanism/s involved are poorly characterized, changes in opioid receptor expression could be a relevant feature. Using a mouse model of postoperative pain, we assessed the expression of MOR and delta opioid receptors (DORs) and the efficacy of Herpes Simplex vector-mediated proenkephalin release (SHPE) preventing postoperative nociceptive sensitization induced by remifentanil or surgical incision. We determined MOR and DOR expressions in the dorsal root ganglia and the spinal cord after remifentanil or surgery in CD1 mice, using real-time PCR and Western blotting. We also assessed the effect of SHPE on nociception induced by remifentanil, surgery, and their combination (2 and 7 days after manipulation), using thermal and mechanical tests. Both remifentanil and surgery decreased DOR mRNA levels (up to days 2 and 4, respectively) in the dorsal root ganglia, but not in the spinal cord. No changes were observed in MOR mRNA, or in receptor-protein levels (Western) of either receptor. Pre-treatment with SHPE 7 days before manipulation prevented remifentanil-induced thermal hyperalgesia and mechanical allodynia and the increase in incisional pain observed when surgery was performed under remifentanil anesthesia. SHPE also prevented surgically induced allodynia but not hyperalgesia, which was blocked by the additional administration of RB101, an enkephalinase inhibitor. The study suggests that down-regulation of DOR contributes to remifentanil and surgery-induced nociception, and that postoperative pain is completely reversed by increasing enkephalin levels in the spinal cord and the periphery.

Tolerance to the antinociceptive and antiexudative effects of morphine in a murine model of peripheral inflammation.

Opioids are used in humans in the management of chronic osteoarticular pains, but the development of tolerance to the analgesic effects after continuous administration is still not well understood. Our aim was to characterize morphine tolerance in a murine model of arthritis that mimics the sequence of events occurring in humans. Inflammation was induced by the intraplantar injection of complete Freund's adjuvant (CFA) and tolerance by the implantation of a 75-mg morphine pellet. We assessed the antihyperalgesic (plantar and Randall-Selitto tests), antiallodynic (Von Frey test), and antiexudative (Evans blue) effects of morphine, the mu-opioid receptor (MOR) mRNA levels in dorsal root ganglia (DRG), and MOR protein levels in DRG and plantar tissue. Inflammation induced plasma extravasation, and it significantly increased the antihyperalgesic effects of morphine (p < 0.05). Morphine pellet implantation decreased morphine potency in all tests. ED(50) values decreased 4.4 and 7.3 times in the absence and presence of inflammation in the plantar test and 2.7 and 5.3 times in the Randall-Selitto test, whereas plasma extravasation decreased 4.2 times. MOR mRNA levels in the DRG were not affected 7 days after inflammation, whereas chronic morphine administration induced a discrete increase (p < 0.05). MOR protein in the DRG or the paw was unchanged. The results show that inflammation enhances the development of tolerance to the antihyperalgesic and antiexudative effects of morphine. At the molecular level, our results suggest that these effects are not mediated by changes in MOR expression but by other changes in receptor activation/internalization.

Inhibition of mitochondrial respiration by nitric oxide: its role in glucose metabolism and neuroprotection.

There is an increasing body of evidence demonstrating that inhibition of cytochrome c oxidase by nitric oxide (NO) may be one more step in a signaling cascade involved in the physiologic regulation of cell functions. For example, in both astrocytes and neurons the inhibition of mitochondrial respiration by endogenously produced NO induces transient and modest decreases in cellular ATP concentrations. This mitochondrial impairment may serve as a cellular sensor of energy charges, hence modulating metabolic pathways, such as glycolysis, through AMP-activated protein kinase (AMPK) in astrocytes. In neurons, the NO derivative peroxynitrite anion triggers signaling pathways leading to glucose oxidation through the pentose-phosphate pathway to form reducing equivalents in the form of NADPH. The modulation of these metabolic pathways by nitric oxide or its derivatives may be important for understanding the mechanisms by which this free radical affects neuronal death or survival.

Regulation of glucose metabolism by nitrosative stress in neural cells.

Following brain inflammatory stimuli, astrocytes actively synthesize nitric oxide and peroxynitrite. These nitrogen-derived species trigger a repertoire of biochemical effects, including alteration of mitochondrial function and redox status both in astrocytes and neighboring neurons. Furthermore, under such nitrosative stress astrocytes show remarkable resistance in spite of having their mitochondria impaired, whereas the neighboring neurons show vulnerability. In this review, we discuss recent evidence strongly suggesting that nitrogen-derived species modulate key regulatory steps of glucose metabolism. These involve up-regulation of high-affinity glucose transporter, stimulation of glycolysis at 6-phosphofructo-1-kinase, and activation of pentose-phosphate pathway at glucose-6-phosphate dehydrogenase. We conclude that the orchestrated stimulation of glucose-metabolising pathways by nitric oxide would be a transient attempt of certain neural cells to compensate for the impaired energy status and oxidised glutathione and thus emerge from an otherwise neuropathological outcome.