Antonio Micali

Long term treatment with sodium hyaluronate-containing artificial tears reduces ocular surface damage in patients with dry eye

Background/aims: Several studies have reported that sodium hyaluronate is able to improve both symptoms and signs in patients with dry eye but none have demonstrated an improvement of conjunctival epithelial cell abnormalities of the ocular surface. The aim of this study was to explore the effect of sodium hyaluronate-containing eye drops on the ocular surface of patients with dry eye during long term treatment. Methods: A randomised double blind study was undertaken in 86 patients with medium to severe dry eye (that is, rose bengal and/or fluorescein test score of at least 3, tear film break up time <10 seconds, or Schirmers test <5.5 mm). Patients were treated with either preservative-free sodium hyaluronate or saline for 3 months at a dose of one drop 4–8 times a day. Bulbar impression cytology, slit lamp examinations, and subjective symptoms were evaluated after 1, 2, and 3 months. Impression cytology was considered the primary efficacy parameter of the study. Results: The efficacy analysis was performed on a total of 44 patients who were able to fully adhere to the protocol. After 3 months of treatment sodium hyaluronate improved impression cytology score (p = 0.024 v baseline). At the same time also the difference with respect to placebo was statistically significant (p = 0.036). Study medication was well tolerated and no treatment related adverse events occurred during the study. Conclusions: Sodium hyaluronate may effectively improve ocular surface damage associated with dry eye syndrome.

Beneficial effects of peroxynitrite decomposition catalyst in a rat model of splanchnic artery occlusion and reperfusion

The aim of the present study was to investigate the protective effect of the peroxynitrite decomposition catalyst 5,10,15,20‐tetrakis(2,4,6‐tri‐methyl‐3,5‐disulfonatophenyl)‐porphyrinato iron (III) (FeTMPS) in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite‐induced oxidative processes) in the plasma of the SAO‐shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunore‐activity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO‐shocked rats developed a significant increase of tissue myeloperoxidase and malon‐aldehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 h after reperfusion). Reperfused ileum tissue sections from SAO‐shocked rats showed positive staining for P‐selectin localized mainly in the vascular endothelial cells. Ileum tissue sections obtained from SAO‐shocked rats and stained with antibody to ICAM‐1 showed a diffuse staining. Administration of FeT‐MPS significantly reduced ischemia/reperfusion injury in the bowel, and reduced lipid and the production of peroxynitrite during reperfusion. Treatment with PN catalyst also markedly reduced the intensity and degree of P‐selectin and ICAM‐1 staining in tissue sections from SAO‐shocked rats and improved survival. Our results clearly demonstrate that per‐oxynitrite decomposition catalysts exert a protective effect in SAO and that this effect may be due to inhibition of the expression of adhesion molecules and the tissue damage associated with peroxynitrite‐related pathways.—Cuzzocrea, S., Misko, T. P., Costantino, C., Mazzon, E., Micali, A., Caputi A. P., Macarthur, H., Salvemini, D. Beneficial effects of peroxynitrite decomposition catalyst in a rat model of splanchnic artery occlusion and reperfusion. FASEB J. 14, 1061–1072 (2000)

Beneficial effects of melatonin in a rat model of splanchnic artery occlusion and reperfusion

The aim of the present study was to investigate the protective effect of the pineal secretary product melatonin in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, animals were sacrificed for tissue histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite‐induced oxidative processes) in the plasma of the SAO‐shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO‐shocked rats developed a significant increase of tissue myeloperoxidase and malondialdehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 hr after reperfusion). Reperfused ileum tissue sections from SAO‐shocked rats showed positive staining for P‐selectin, which was mainly localized in the vascular endothelial cells. Ileum tissue sections obtained from SAO‐shocked rats with anti‐intercellular adhesion molecule (ICAM‐1) antibody showed a diffuse staining. Melatonin (applied at 3 mg/kg, 5 min prior to reperfusion, followed by an infusion of 3 mg/kg per hr), significantly reduced ischemia/reperfusion injury in the bowel as evaluated by histological examination. This prevented the infiltration of neutrophils into the reperfused intestine, as evidenced by reduced myeloperoxidase activity and reduced lipid peroxidation. This was evaluated by malondialdehyde activity which reduced the production of peroxynitrite during reperfusion, markedly reduced the intensity and degree of P‐selectin and ICAM‐1 in tissue section from SAO‐shocked rats and improved their survival. Taken together, our results clearly demonstrate that melatonin treatment exerts a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite‐related pathways and subsequent reduction of neutrophil‐mediated cellular injury.

Impression cytology of the conjunctival epithelium in patients with vernal conjunctivitis.

The alterations in the conjunctival epithelium during the course of vernal conjunctivitis were examined by conjunctival impression cytology. The study was carried out on 53 patients with vernal conjunctivitis and 20 normal subjects as control. The results of impression cytology demonstrated that all cytological parameters were significantly modified in vernal conjunctivitis patients; the earliest alterations were found in the distribution of goblet cells, in the intercellular junctions, in the chromatin morphology and in the degree of keratinisation. The morphometric comparison showed that in vernal conjunctivitis patients the mean number of goblet cells per field was significantly higher than in controls. Moreover the mean diameter of goblet cells was smaller in patients than in controls. Impression cytology can, therefore, be a simple, non-invasive and cheap method for the study of the ocular surface in vernal conjunctivitis.

Hyaluronan reduces inflammation in experimental arthritis by modulating TLR-2 and TLR-4 cartilage expression.

Previous studies have reported that low molecular mass HA and highly polymerized HA respectively elicited pro- and anti-inflammatory responses by modulating the toll-like receptor 4 (TLR-4) and the TLR-2. The activation of TLR-4 and TLR-2 mediated by collagen-induced arthritis (CIA) induces the myeloid differentiation primary response protein (MyD88) and the tumor necrosis factor receptor-associated factor 6 (TRAF6), and ends with the liberation of NF-kB which, in turn, stimulates pro-inflammatory cytokine production. The aim of this study was to investigate the influence of high molecular weight HA at different concentrations on TLR-4 and TLR-2 modulation in CIA in mice. Arthritis was induced in mice via intradermal injection of an emulsion containing bovine type II collagen in complete Freunds adjuvant. Mice were treated with HA intraperitoneally daily for 30days. CIA increased TLR-4, TLR-2, MyD88 and TRAF6 mRNA expression and the related protein in the cartilage of arthritic joints. High levels of both mRNA and related protein were also detected for tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL-1-β), interleukin-17 (IL-17), matrix metalloprotease-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in the joint of arthritic mice. HA treatment significantly limited CIA incidence and decreased all the parameters up-regulated by CIA. The improvement of biochemical parameters was also supported by histological analysis, plasma and synovial fluid HA levels. These results suggest that the TLR-4 and TLR-2 play an important role in the arthritis mechanism and the interaction/block of HA at high molecular mass may reduce inflammation and cartilage injury.

The protective role of endogenous glutathione in carrageenan-induced pleurisy in the rat

In the present study we investigated the protective role of endogenous glutathione, a known free radical scavenger, in rats subjected to carrageenan-induced pleurisy. In vivo depletion of endogenous glutathione pools with L-buthionine-(S,R)-sulfoximine (BSO, 1 g/kg for 24 h, intraperitoneally) enhances the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase activity and lipid peroxidation were significantly increased in BSO pretreated rats. However, the inducible nitric oxide (NO) synthase in lung samples was unaffected by BSO pretreatment. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from carrageenan-treated rats, which was massively enhanced by BSO pretreatment. Furthermore, in vivo BSO pretreatment significantly increased peroxynitrite formation as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, enhanced the appearance of DNA damage, the decrease in mitochondrial respiration and partially decreased the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. In vivo treatment with exogenous glutathione (50 mg/kg i.p.) significantly reverts the effects of BSO and exerts anti-inflammatory effects. Thus, endogenous glutathione plays an important protective role against carrageenan-induced local inflammation.

Polydeoxyribonucleotide reduces cytokine production and the severity of collagen‐induced arthritis by stimulation of adenosine A2A receptor

OBJECTIVE Broad antiinflammatory effects following adenosine A(₂A) receptor stimulation have been demonstrated in acute inflammatory diseases, including arthritis. Polydeoxyribonucleotide (PDRN) activates the adenosine A(₂A) receptor. This study was undertaken to investigate the effects of PDRN in collagen-induced arthritis (CIA) in mice. METHODS Arthritis was induced in DBA/1 mice by an intradermal injection of 100 μl of bovine type II collagen in Freunds complete adjuvant. Mice were immunized a second time 21 days later. Control animals received 100 μl of a saline solution. Animals with CIA were randomized to receive one of the following: vehicle (1 ml/kg); PDRN (8 mg/kg intraperitoneally daily); 3,7-dimethyl-propargylxanthine (DMPX), a specific adenosine A(₂A) receptor antagonist (0.1 mg/kg intraperitoneally daily); or PDRN plus DMPX. The treatment was initiated immediately after the second immunization and continued to day 45. Clinical evaluation of arthritis was performed throughout the study. On day 45, the animals were killed and the severity of arthritis was evaluated histologically. Cartilage expression and circulating levels of high mobility group box chromosomal protein 1 (HMGB-1), tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and IL-10 were investigated. Inflammatory cytokine production was also evaluated in stimulated human chondrocytes treated with PDRN. RESULTS PDRN treatment significantly ameliorated clinical signs of arthritis, improved histologic damage, reduced the cartilage expression and circulating levels of HMGB-1, TNFα, and IL-6, and enhanced IL-10 expression. The concomitant administration of DMPX and PDRN ablated the PDRN-induced protective effect in experimental arthritis. PDRN also reduced cytokine production from stimulated human chondrocytes. CONCLUSION Our findings indicate that PDRN may represent a new alternative for the treatment of arthritis.

Morphological changes of the conjunctival epithelium in contact lens wearers evaluated by impression cytology

Purpose Conjunctival changes induced by rigid, gas-permeable and soft contact lenses (CL) were investigated using impression cytology. A scoring system based on seven parameters (specimen cellularity, cell-to-cell contacts, nucleus/cytoplasm ratio, chromatin, goblet cell distribution, keratinisation, inflammatory cells) was used to evaluate the morphological results.Methods One hundred and seventy-two CL-wearing eyes and 40 control eyes were examined. The population was divided into three groups: group 1, control eyes; group 2, asymptomatic CL-wearing eyes; group 3, CL-wearing eyes with intolerance problems. Impression cytology was carried out and all specimens were evaluated according to a scoring system.Results Group 1 specimens always showed normal features in the conjunctival epithelium. In group 2, rigid and gas-permeable lenses produced greater changes in conjunctival morphology than soft lenses, especially as regards cellularity, nucleus/ cytoplasm ratio, goblet cell distribution and total score. On the contrary, in group 3 wearers of soft lenses showed higher partial and total scores than wearers of gas-permeable and rigid lenses.Conclusions In asymptomatic CL wearers conjunctival morphology was better preserved in wearers of soft lenses. In patients with intolerance problems, wearers of soft lenses showed the worst cytological features. Impression cytology, evaluated with this scoring system, can be used to demonstrate epithelial damage occurring in CL-wearing patients.

ROS-Mediated NLRP3 Inflammasome Activation in Brain, Heart, Kidney, and Testis Ischemia/Reperfusion Injury

Ischemia and reperfusion (I/R) causes a reduction in arterial blood supply to tissues, followed by the restoration of perfusion and consequent reoxygenation. The reestablishment of blood flow triggers further damage to the ischemic tissue through reactive oxygen species (ROS) accumulation, interference with cellular ion homeostasis, and inflammatory responses to cell death. In normal conditions, ROS mediate important beneficial responses. When their production is prolonged or elevated, harmful events are observed with peculiar cellular changes. In particular, during I/R, ROS stimulate tissue inflammation and induce NLRP3 inflammasome activation. The mechanisms underlying the activation of NLRP3 are several and not completely elucidated. It was recently shown that NLRP3 might sense directly the presence of ROS produced by normal or malfunctioning mitochondria or indirectly by other activators of NLRP3. Aim of the present review is to describe the current knowledge on the role of NLRP3 in some organs (brain, heart, kidney, and testis) after I/R injury, with particular regard to the role played by ROS in its activation. Furthermore, as no specific therapy for the prevention or treatment of the high mortality and morbidity associated with I/R is available, the state of the art of the development of novel therapeutic approaches is illustrated.

Relaxin improves multiple markers of wound healing and ameliorates the disturbed healing pattern of genetically diabetic mice.

Diabetic mice are characterized by a disrupted expression pattern of VEGF (vascular endothelial growth factor), and impaired vasculogenesis during healing. Experimental evidence suggests that RLX (relaxin) can improve several parameters associated with wound healing. Therefore we investigated the effects of porcine-derived RLX in diabetes-related wound-healing defects in genetically diabetic mice. An incisional wound model was produced on the back of female diabetic C57BL/KsJ-m+/+Leptdb (db+/db+) mice and their normal littermates (db+/+m). Animals were treated daily with porcine RLX (25 μg/mouse per day, subcutaneously) or its vehicle. Mice were killed on 3, 6 and 12 days after skin injury for measurements of VEGF mRNA and protein synthesis, SDF-1α (stromal cell-derived factor-1α) mRNA and eNOS (endothelial NO synthase) expression. Furthermore, we evaluated wound-breaking strength, histological changes, angiogenesis and vasculogenesis at day 12. Diabetic animals showed a reduced expression of VEGF, eNOS and SDF-1α compared with non-diabetic animals. At day 6, RLX administration resulted in an increase in VEGF mRNA expression and protein wound content, in eNOS expression and in SDF-1α mRNA. Furthermore, the histological evaluation indicated that RLX improved the impaired wound healing, enhanced the staining of MMP-11 (matrix metalloproteinase-11) and increased wound-breaking strength at day 12 in diabetic mice. Immunohistochemistry showed that RLX in diabetic animals augmented new vessel formation by stimulating both angiogenesis and vasculogenesis. RLX significantly reduced the time to complete skin normalization and this effect was abrogated by a concomitant treatment with antibodies against VEGF and CXCR4 (CXC chemokine receptor 4), the SDF-1α receptor. These data strongly suggest that RLX may have a potential application in diabetes-related wound disorders.