Antonina Pisani

Protective effect of glutathione on kainic acid-induced neuropathological changes in the rat brain

Abstract 1. 1. Glutathione (GSH), injected by slow intravenous (i.v.) infusion (7.9 μl/min, for 4 hr; total dose: 1.5 g/kg), starting 10 min after i.v. injection of kainic acid (KA; 12 mg/kg) in the rat reduced the decrease in local cerebral glucose utilization observed 48 hr following the administration of the neurotoxin. 2. 2. Furthermore, it blocked the neuronal loss in hippocampal CA1 and CA3 regions, and prevented, in the hippocampus, the development of edema and the marked depletion in the endogenous brain GSH pool. 3. 3. One can speculate that this protective effect of exogenous GSH is correlated to its capacity to scavenge free radicals, thus preventing the accumulation of oxidant chemical species and the consequent reduction of cellular antioxidant defense.

Matrix Metalloproteinase 9 and Transglutaminase 2 Expression at the Ocular Surface in Patients with Different Forms of Dry Eye Disease

OBJECTIVE To evaluate the expression of matrix metalloproteinase 9 (MMP9) and transglutaminase 2 (TG2) in different forms of dry eye. DESIGN Case control study. PARTICIPANTS Seventy-five female subjects divided into 3 groups: group 1, 15 healthy controls; group 2, 30 subjects with Sjögren syndrome (SS); and group 3, 30 subjects with Meibomian gland dysfunction (MGD). METHODS A clinical assessment was carried out and impression cytologic specimens were processed for immunoperoxidase staining for MMP9 and TG2 and real-time polymerase chain reaction analyses were carried out for MMP9, TG2, interleukin-6, interferon-γ, B-cell lymphoma 2, and caspase 3. To study MMP9 and TG2 expression after anti-inflammatory treatment, patients were divided into 2 subgroups, one treated with saline and the other treated with saline plus topical corticosteroid eye drops (0.5% loteprednol etabonate) 4 times daily for 15 days. For statistical analysis, Student t test, Mann-Whitney U test, and Spearmans correlation coefficient were used as appropriate. MAIN OUTCOME MEASURES Conjunctival expression of MMP9 and TG2. RESULTS MMP9 and TG2 expression were higher in both patient groups than in controls (P < 0.0001). Group 2 patients showed higher expression than group 3 (P < 0.0001). The Spearmans correlation coefficient showed in group 2 a positive correlation between MMP9 and TG2 expression (ρ = 0.437; P = 0.01), but no correlation in group 3 (ρ = 0.143; P = 0.45). Corticosteroid treatment significantly reduced MMP9 and TG2 expression in both groups, ameliorating symptoms and signs. A much higher percentage reduction was observed in SS. CONCLUSIONS The pathogenic mechanisms of the 2 forms of dry eye give an account for the different MMP9 and TG2 expressions in the 2 groups of patients. The higher expression in SS is determined by the direct autoimmune insult to the ocular surface epithelia, whereas in MGD patients, with an epithelial damage due to an unbalanced tear secretion, the molecules expression is significantly lower, although higher than in controls. The corticosteroid treatment induced a reduction of both molecules, although higher in SS than in MGD, because of its direct inhibitory effect on inflammation.

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.

Morphologic and Confocal Investigation on Salzmann Nodular Degeneration of the Cornea

PURPOSE To investigate the ultrastructure of advanced Salzmann nodular degeneration (SND) and to correlate it to confocal in vivo findings. METHODS The corneal degenerative nodules from four patients with SND were examined with confocal microscopy and then removed and processed for light microscopy (LM) and transmission electron microscopy (TEM). RESULTS The confocal examination revealed elongated basal epithelial cells and activated keratocytes in the anterior stroma near the nodules. Occasional subbasal nerves and tortuous stromal nerve bundles were observed. With LM and TEM, five zones were described: one internodular and four pertaining the nodule, each characterized by peculiar aspects of the epithelium and stroma. As also confirmed by the morphometry, in the zones corresponding to the nodules, the epithelium was lower and with fewer cell layers than the peripheral zones. Over the nodules, the basement membrane was discontinuous or absent and the Bowmans layer, when present, had a granular-filamentous appearance. The nodular stroma was formed by many activated keratocytes and irregular lamellae. Subbasal nerves were always isolated and had degenerative changes in the center of the nodule. CONCLUSIONS This work illustrates the confocal microscopic findings associated with LM and TEM observations in patients with advanced SND. Our data revealed milder changes of the epithelium together with more pronounced changes of the basement membrane and Bowmans layer, which are aspects of increased keratocyte activity and an altered nerve pattern. All of these structures seem to contribute to the characteristic corneal disorganization of SND.

Ultrastructural Study of the Conjunctival Epithelium in the Mongolian Gerbil (Meriones unguiculatus)

The conjunctival epithelium of the Mongolian gerbil (Meriones unguiculatus) was studied using the transmission and scanning electron microscopy. The superficial cells of the conjunctival epithelium were isoprismatic in shape and were covered, on their luminal surface, with microplicae. They were connected with the adjacent cells by junctional complexes and desmosomes. Some of the superficial cells were partially or completely detached from the underlying intermediate cells: this morphological pattern was interpreted as an expression of cellular renewal of the conjunctival surface. Goblet cells were either isolated or gathered in clusters: in any case, they were firmly connected with the adjacent epithelial cells. The apical part of the goblet cells was covered with a great number of long microvilli: they showed a variety of morphological aspects, which were interpreted as occurring in a sequential pattern during the secretory process. The Mongolian gerbil can be considered among the laboratory animals used for ophthalmic research.

Flavocoxid Protects Against Cadmium-Induced Disruption of the Blood–Testis Barrier and Improves Testicular Damage and Germ Cell Impairment in Mice

Cadmium (Cd) causes male infertility. There is the need to identify safe treatments counteracting this toxicity. Flavocoxid is a flavonoid that induces a balanced inhibition of cyclooxygenase (COX)-1 and COX-2 peroxidase moieties and of 5-lipoxygenase (LOX) and has efficacy in the male genitourinary system. We investigated flavocoxid effects on Cd-induced testicular toxicity in mice. Swiss mice were divided into 4 groups: 2 control groups received 0.9% NaCl (vehicle; 1 ml/kg/day) or flavocoxid (20 mg/kg/day ip); 2 groups were challenged with cadmium chloride (CdCl2; 2 mg/kg/day ip) and administered with vehicle or flavocoxid. The treatment lasted for 1 or 2 weeks. The testes were processed for biochemical and morphological studies. CdCl2 increased phosphorylated extracellular signal-regulated kinase (p-ERK) 1/2, tumor necrosis factor (TNF)-α, COX-2, 5-LOX, malondialdehyde (MDA), B-cell-lymphoma (Bcl)-2-associated X protein (Bax), follicle-stimulating hormone (FSH), luteinizing hormone (LH), transforming growth factor (TGF) -β3, decreased Bcl-2, testosterone, inhibin-B, occludin, N-Cadherin, induced structural damages in the testis and disrupted the blood-testis barrier. Many TUNEL-positive germ cells and changes in claudin-11, occludin, and N-cadherin localization were present. Flavocoxid administration reduced, in a time-dependent way, p-ERK 1/2, TNF-α, COX-2, 5-LOX, MDA, Bax, FSH, LH, TGF-β3, augmented Bcl-2, testosterone, inhibin B, occludin, N-Cadherin, and improved the structural organization of the testis and the blood-testis barrier. Few TUNEL-positive germ cells were present and a morphological retrieval of the intercellular junctions was observed. In conclusion, flavocoxid has a protective anti-inflammatory, antioxidant, and antiapoptotic function against Cd-induced toxicity in mice testis. We suggest that flavocoxid may play a relevant positive role against environmental levels of Cd, otherwise deleterious to gametogenesis and tubular integrity.

Phenotype-genotype correlation in patients with Schnyder corneal dystrophy.

Purpose: The aim of this study was to analyze the corneal morphology features and define mutations in the UbiA prenyltransferase domain–containing 1 (UBIAD1) gene in patients with Schnyder corneal dystrophy from a Polish population. Methods: Five affected and 15 unaffected members originating from 3 families with Schnyder corneal dystrophy were included in the study. Phenotype analysis consisted of visual acuity, slit-lamp biomicroscopy with photography, time domain optical coherence tomography, spectral domain optical coherence tomography, and confocal microscopy. Three patients underwent a penetrating keratoplasty. Corneal buttons obtained from the penetrating keratoplasty were processed for light microscopy. Results: A novel mutation I245N of the UBIAD1 gene was revealed in 1 proband and associated with the phenotype without central corneal opacities. The analysis of the other patients showed the N102S mutation. In vivo corneal morphology analysis using optical coherence tomography and confocal microscopy confirmed the presence of multiple crystalline corneal deposits in all affected corneas. The histological examination revealed multiple empty widenings of the corneal lamellae that could represent lipids removed from the specimen. Conclusions: N102S may also be a mutation hotspot in the Polish population, as in other previously reported populations. Corneal crystals formed a characteristic pattern on optical coherence tomography scans.

The SOD mimic MnTM-2-PyP(5+) reduces hyaluronan degradation-induced inflammation in mouse articular chondrocytes stimulated with Fe (II) plus ascorbate

In pathological conditions, oxidative burst generates hyaluronan (HA) fragmentation with a consequent increase in the number of small HA oligosaccharides. These fragments are able to stimulate an inflammatory response in different cell types by activating the CD44 and the toll-like receptors 4 (TLR-4) and 2 (TLR-2). The stimulation of CD44 and TLRs in turn activates the NF-kB which induces the production of several pro-inflammatory mediators that amplify and perpetuate inflammation. We aimed to study the antioxidant effect of the SOD mimic, synthetic manganese porphyrin, Mn(III) 5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin (MnTM-2-PyP(5+)) on preventing HA degradation in mouse articular chondrocytes stimulated with Fe (II) plus ascorbate. Fe (II) plus ascorbate stimulation induced oxidative burst confirmed by high levels of hydroxyl radical/peroxynitrite production, increased lipid peroxidation and HA degradation. HA fragments highly induced mRNA expression and the related protein production of CD44, TLR-4 and TLR-2, NF-kB activation and significantly up-regulated the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and other pro-inflammatory mediators, i.e. matrix metalloprotease 13 (MMP-13) and inducible nitric oxide synthase (iNOS). Treatment of cells with MnTM-2-PyP(5+)was able to attenuate oxidative burst, HA degradation and NF-kB activation, and markedly decreased mRNA expression of CD44, and TLRs and the related protein synthesis, as well as the levels of up-regulated inflammatory mediators. Adding a specific HA-blocking peptide (PEP-1) to cells significantly reduced all the inflammatory parameters up-regulated by Fe (II) plus ascorbate, and increased MnTM-2-PyP(5+) activity. These findings suggest that HA degradation plays a key role in the initial inflammatory response of cartilage and antioxidants and could be a useful tool to prevent the propagation of this mechanism.

Cadmium, organ toxicity and therapeutic approaches. A review on brain, kidney and testis damage

BACKGROUND Cadmium (Cd) is a heavy metal particularly hazardous for human health, as it is highly diffused and, therefore, a ubiquitous environmental toxicant. In fact, in the general population, the main sources of exposure are food, cigarette smoking, inhalation of ambient air, drinking water, contaminated soil or dust. Furthermore, an occupational exposure usually involves human during mining, fume inhalation or manufacturing nickel-cadmium battery, electroplating and paint pigments that utilize Cd. METHODS We undertook a structured search in literature about Cd. This metal is noxious on the cells of many organs, among which the kidney, the testis and the brain will be considered in this review. RESULTS The toxic effects induced by Cd include many specific mechanisms, such as the oxidative stress, cellular death and inflammation. As no specific therapy for the prevention or treatment of the morbidity and mortality associated with Cd exposure is available, the state of the art of the therapeutic approaches is illustrated. CONCLUSION Nowadays, a therapy able to counteract Cd toxicity is still lacking and the development of new therapeutic agents is requested.

NLRP3 Inflammasome Involvement in the Organ Damage and Impaired Spermatogenesis Induced by Testicular Ischemia and Reperfusion in Mice

We investigated the role of the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome during testis ischemia and reperfusion injury (TI/R) in wild-type (WT) and NLRP3 knock-out (KO) mice. WT and KO mice underwent 1 hour testicular ischemia followed by 4 hours and 1 and 7 days of reperfusion or a sham TI/R. Furthermore, two groups of WT mice were treated at the beginning of reperfusion and up to 7 days with two inflammasome inhibitors, BAY 11-7082 (20 mg/kg i.p.) or Brilliant Blue G (45.5 mg/kg i.p.), or vehicle. Animals were killed with a pentobarbital sodium overdose at 4 hours and 1 and 7 days, and bilateral orchidectomies were performed. Biochemical and morphologic studies were carried out in all groups. TI/R in WT mice significantly increased caspase-1 and interleukin (IL)-1β mRNA after 4 hours and IL-18 mRNA at 1 day of reperfusion (P ≤ 0.05). There was also a significant increase in caspase-3 and terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling–positive cells, marked histologic damage, and altered spermatogenesis in WT mice in both testes after 1 and 7 days of reperfusion. KO TI/R mice, WT TI/R BAY 11-7082, and Brilliant Blue G treated mice showed a significant reduced IL-1β and IL-18 mRNA expression, blunted caspase-1 and -3 expression, minor histologic damages, low terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling activity, and preserved spermatogenesis. These data suggest that the activation of NLRP3 plays a key role in TI/R, and its inhibition might represent a therapeutic target for the management of patients with unilateral testicular torsion.