Degenerative events in retina and optic nerve induced by inhibition of carnitine transport.

Biochemical and pharmacological evidence supports the hypothesis that the mechanism of action of mildronate [3-(2,2,2-trimethylhydrazinium)propionate dihydrate] is based on its regulatory effect on carnitine concentration. The present study demonstrates that carnitine acts as a neuroprotective agent both in optic nerve head and in retinal ganglion cell (RGC) by means of antioxidant and antiradical activities. In fact, carnitine normalized the increase in caspase-3, cellular apoptosis susceptibility protein (CAS) and inducible nitric oxide synthase (iNOS) expression by stabilizing mitochondrial membranes, as assessed by quantitative and qualitative analysis. This research shows that the neuroprotective effects of carnitine result, at least partially, from anti-neurodegenerative (anti-apoptotic) and anti-inflammatory mechanisms. It is suggested that the molecular conformation of carnitine can facilitate its easy binding to mitochondria, and regulate the expression of different signal molecules, hence maintaining normal cellular signaling and survival by modulating caspase-3 activity.

Mechanisms of ocular neuroprotection by antioxidant molecules in animal models.

This work was conducted to evaluate the efficacy of a treatment on retinal ganglion cells (RGC) and on astrocytes of the optic nerve of glaucomatous eyes, using a combination of alpha-lipoic acid (ALA) and superoxide dismutase (SOD). Thirty-two male Wistar rats were fed with a diet supplemented with ALA, SOD, ALA and SOD or with no product for 8 weeks. Ocular hypertension was induced with 2% methylcellulose (MTC) and then rats were sacrificed. TUNEL assay showed a marked fluorescence in the ganglion cells and astrocytes of MTC-treated rats evidencing induction of apoptosis. In contrast, sections of eyes pretreated with ALA and SOD showed a lack of fluorescence quite similar to that of the controls. Similarly, eyes sections from rats pre-treated with ALA and SOD showed reduced differential expression of inducible nitric oxide synthase (iNOS) and of caspase-3 in compared to normally-fed/MTC-inoculated cases. An increase of ALA and SOD exerts an antiapoptotic effect and protects against oxidative stress and hence against the structural remodelling of the RGCs and astrocytes of the optic nerve in the presence of an ischemic and pressure stress.

Oxidative stress in the closed-eyelid test: management of glaucoma.

BACKGROUND AND OBJECTIVES: To evaluate the role of antioxidant drugs in the tonometric increase that follows the closed eyelid test (CET), a predicitive test for glaucoma, after administration of antioxidant substances was observed. MATERIALS AND METHODS: 30 subjects of 54.57+/=5.62 years, 13 males and 17 females, were examined by measuring the ocular pressure after 1 hour from the CET, both in normal conditions and after the administration of antioxidants such as: vitamin A (50,000 IU/die), vitamin E (600 mg/die), and vitamin C (1000 mg/die). The increases in temperature of the iridocorneal angle and of the iris were also measured in the same conditions with an infrared Thermo-Precision tonometer (Sola Electro-Optics, China) both before and after CET. RESULTS: The results showed increased pressure after CET and decreased pressure after the administration of each antioxidant substance, although vitamin A was found to be more effective and with statistically significant values compared to vitamins E and C. CONCLUSIONS: Considering the responses obtained after administration of antioxidant drugs, the ocular hypertension induced after CET could be a response to mixed stress, oxidative and thermic, with degenerative effects on the trabecular meshwork (TM). Besides, in light of these considerations the research results underline that the open angle glaucoma (OAG) should be considered a multifactorial degenerative disease.

Topotecan hydrochloride effects on retinal vessels in newborn rats.

A physiological system, i.e. rodent retina during vessel formation and hierarchical organization, was utilised for assaying antiangiogenic properties of Topotecan, a topoisomerase I inhibitor, capable of inhibiting tumoral growth in animal models of retinoblastoma. In particular we analysed possible differences in effectiveness and side effects among different drug dosages and ways of administration. In the present research only qualitative analyses were undertaken. After preliminary experiments, in which suckling animals subcutaneously treated with Topotecan dosages comprised between 9 and 3 mg/kg underwent high lethality and extremely severe systemic damages, 7 day-old rats were subcutaneously, intravenously or peribulbary injected with a single dose of 1 mg/kg; retinal vessels were visualized in retinal fluorangio-graphies taken 1 and 2 weeks after treatment. The most important and frequent alterations were found to affect radial vessels, which showed non-perfused and/or regionally mislocated segments, together with abnormal branching and enlargements in retinal periphery; persistence of capillary-free periarteriolar regions, non-vascularised regions and spots of extravascular FITC were also detected. Despite the high individual variability the alterations were substantially similar among the different ways of drug administration, while they appeared milder in 21 day-old rats, with respect to younger ones. The extensive vascular remodelling found after Topotecan administration, besides demonstrating the antiangiogenic properties of this chemioterapic drug, confirms the rodent retina as a highly valuable model system for studying angiogenesis modulation.

Degenerative effects in rat eyes after experimental ocular hypertension.

This study was used to evaluate the degenerative effects on the retina and eye-cup sections after experimental induction of acute ocular hypertension on animal models. In particular, vascular events were directly focused in this research in order to assess the vascular remodeling after transient ocular hypertension on rat models. After local anaesthesia by administration of eye drops of 0.4% oxibuprocaine, 16 male adult Wistar rats were injected in the anterior chamber of the right eye with 15 µL of methylcellulose (MTC) 2% in physiological solution. The morphology and the vessels of the retina and eye-cup sections were examined in animals sacrificed 72 h after induction of ocular hypertension. In retinal fluorescein angiographies (FAGs), by means of fluorescein isothiocyanate-coniugated dextran (FITC), the radial venules showed enlargements and increased branching, while the arterioles appeared focally thickened. The length and size of actually perfused vessels appeared increased in the whole superficial plexus. In eye-cup sections of MTC-injected animals, in deep plexus and connecting layer there was a bigger increase of vessels than in controls. Moreover, the immunolocalization of astrocytic marker glial fibrillary acidic protein (GFAP) revealed its increased expression in internal limiting membrane and ganglion cell layer, as well as its presence in Müller cells. Finally, the pro-angiogenic factor vascular endothelial growth factor (VEGF) was found to be especially expressed by neurones of ganglion cell layer, both in control and in MTC-injected eyes. The data obtained in this experimental model on the interactions among glia, vessels and neurons should be useful to evaluate if also in glaucomatous patients the activation of vessel-adjacent glial cells might play key roles in following neuronal dysfunction.

Carnitine reduces the lipoperoxidative damage of the membrane and apoptosis after induction of cell stress in experimental glaucoma.

The pathological damage caused by glaucoma is associated to a high intraocular pressure. The ocular hypertone is most likely due to a defective efflux of aqueous humor from the anterior chamber of the eye. Ocular hypertension causes apoptotic death of retinal ganglion cells and overexpression of molecular markers typical of cell stress response and apoptosis. In this work, we report on the neuroprotective, antiapoptotic and antioxidant action of a natural substance, -carnitine. This compound is known for its ability to improve the mitochondrial performance. We analyze a number of cellular and molecular markers, typical of ocular hypertension and, in general, of the cell stress response. In particular, L-carnitine reduces the expression of glial fibrillary acidic protein, inducible nitric oxide synthase, ubiquitin and caspase 3 typical markers of cell stress. In addition, the morphological analysis of the optic nerve evidenced a reduction of the pathological excavation of the optic disk. This experimental hypertone protocol induces a severe lipoperoxidation, which is significantly reduced by L-carnitine. The overall interpretation is that mortality of the retinal cells is due to membrane damage.

Reduction of cell proliferation induced by PD166866: an inhibitor of the basic fibroblast growth factor.

UNLABELLED: Cell proliferation control plays a key role in tumor development. The basic Fibroblast Growth Factor (bFGF), as well as other growth factors, is involved in several pathologies characterized by dysregulation of cell proliferation. In the present work the effects of PD166866, a very potent and selective tyrosine kinase inhibitor were evaluated. Cultured murine fibroblasts (the cell line 3T6) were used to assess the FGFR-1 inhibition mediated by PD166866. Evaluation of cell viability and molecular biology techniques were adopted. PD166866 controls negatively the bFGF/FGFR-1 system thus promoting a significant reduction of cell proliferation and loss of viability in 3T6 cells. The drug possibly controls proliferation via induction of apoptosis as evidenced by a relevant chromatin degradation. CONCLUSION: This study demonstrated that PD166866 might be used in the control of fibrotic proliferative diseases, as well as in other tumor pathologies.

The diimide drug PIPER has a cytotoxic dose-dependent effect in vitro and inhibits telomere elongation in HELA cells.

It is known that, in vitro, PIPER (N,N'-bis [2-(1-piperidino)ethyl]-3,4,9,10-tetracarboxylic diimide) induces the formation of the Hoogsteen quadruplex structure in telomere DNA, thus inhibiting the polymerisation of telomeric repeats. Since the action of PIPER in vivo has been scarcely investigated, this study was addressed to gain some insight into the effects of this drug on cultured HeLa cells. Vital staining with erythrosine, performed on cells exposed to different PIPER concentrations (from 1 to 50 microM), showed that the drug exerts a dose-dependent cytotoxic effect, clearly evident after a short-term (24 h) treatment. This early cytotoxic effect of PIPER on cultured HeLa cells was confirmed by a spectrophotometric/colorimetric method employing methylthiazoletetrazolium (Mossmann assay). Hematoxylin/eosin staining of cells treated with PIPER for 24 h showed a nuclear condensation and a cytoplasmic vacuolisation, very pronounced at higher drug concentrations. These pictures suggest that PIPER-induced cell death might be of the apoptotic type. Finally, the anti-telomerase activity of PIPER was monitored by TRAP assay, performed on HeLa cell nuclear extracts treated with increasing drug concentrations. It was found that some inhibition of telomerase is apparent even at low concentrations, while at the highest concentration the enzyme is completely inhibited. These results indicate that the cytotoxic power of PIPER is possibly related to its antitelomeric effect.

Ubiquitin dependent proteolysis is activated in apoptotic fibroblasts in culture.

The ubiquitin mediated pathway constitutes an early response in cultured cells where apoptosis, assessed by internucleosomal specific DNA fragmentation, was induced by serum withdrawal. Data demonstrate that nuclear ubiquitin proteolytic system, but not cytoplasmic, is activated. This activation is paralleled by a substantial chromatin de-condensation. We suggest that chromatin relaxation is causative of the fragmentation since it exposes the DNA to nucleolytic attack. Finally, maintenance of homeostasis and induction of apoptosis seem to undergo a parallel contemporary pathway with a possible mutual feedback.

A murine cell culture model for post-trabeculectomy anfibrotic treatment: Induction of apoptosis by Cyclosporin.

PURPOSE: Experimental trials aimed at the research of selective antifibrotic agents are under development for the alternative treatment of glaucoma patients who are usually considered high-risk post-surgical individuals after trabeculectomy. Authors present here an in vitro model system for the treatment of post-trabeculectomy patients. The study is aimed at the evaluation of different drugs in a mouse fibroblast model. METHODS: The antifibrotic activity of Cyclosporin A, Interferon 2alpha, 5-Fluorouracyl was investigated on 3T6 cells in culture. Cell viability and proliferation was assessed after drug treatment. Molecular analysis of DNA degradation was evaluated by means of radioactive labeling and gel electrophoresis. RESULTS: The three drugs were shown to affect cell proliferation and viability in a differential fashion. However, only Cyclosporin A was able to control cell proliferation, inducing apoptosis. This phenomenon was reduced by supplementation of trolox, a compound known to inhibit programmed cell death. These results strongly suggest that this model system might be useful as a test of pharmacological functionality. CONCLUSION: A rapid and efficient model system is described for the assessment of cell viability and proliferation after treatment with agents of potential pharmacological use. Cyclosporin A induces a significant apoptosis. This is important for the negative control of fibrotic degeneration in post-trabeculectomy that is required for successful surgery in glaucoma patients. Therefore, Cyclosporin A might become a clinically interesting drug for the antifibrotic treatment of post-trabeculectomy.

Effect of different whole body hyperthermic sessions on the heat shock response in mice liver and brain.

We examined by Western blots the effect of variations of the heating sessions, such as duration and intensity on the following aspects: 70-kDa heat shock protein (HSP70) and HSP72 induction. Protein ubiquitination PLCgamma , PKCepsilon and PKCalpha levels in murine liver and brain were also studied. Results demonstrated that maximal induction of HSP72 was obtained after heat shock at 43.5 degrees C in both organs. Preconditioning at lower temperatures (either acclimation to 39 degrees C or induction of thermotolerance to 43.5 degrees C with a single exposure to 39 degrees C) attenuated the heat shock response. Hepatic HSP72 induction was elicited only as a consequence of hyperthermia since either fasting or restraint were unable to trigger its synthesis. On the contrary, a ubiquitination decrease of a 31 kDa protein was obtained both after hyperthermia and fasting This indicates that the latter is a more generic response of hepatic cells to noxious stimuli. Analysis of the above mentioned enzymes showed that in liver of naive mice PKCalpha is barely present while PKCepsilon is quite abundant. All hyperthermic treatments caused a general decrease of the latter, except for the heat shock at 43.5 degrees C that caused an increase. PLCgamma decreased after all heating sessions. It is known that hyperthermia in the range of 41-45 degrees C induces apoptotic death in many cell types. Therefore we analyzed the presence of the typical apoptotic DNA ladder. Our data strongly suggest that both hyperthermia and restraint induce necrosis in liver while apoptosis and necrosis become evident in brain. All these effects are still present 24 h from the last heating session: This indicates that in vivo, hyperthermia produces long term modifications of the hepatic cell.

Experimental ocular acute hypertension-induced chromatinic alterations in astrocytic cells in rat optic nerve.

The effects of ocular acute hypertension experimentally induced on the astrocyte cells of rat have been studied. Evaluation was made of the damage to the chromatin of those cells by means of cytochemical (haematoxylin-eosin) analysis and of the state of fragmentation of the DNA by means of the TUNEL technique as well as the protective effect of the peroxide scavenger, troxol, on those events.

Induced acute ocular hypertension: mode of retinal cell degeneration.

The effects of experimental hypertension on retinal cells were studied. Evaluation was made of IOP levels and degree of cell damage by cytochemical and DNA analysis, and degeneration modes: necrosis and apoptosis.

Ubiquitin-mediated stress response in a rat model of brain transient ischemia/hypoxia.

Ubiquitin (Ub) is a small 76-residue protein, involved in intracellular protein degradation through a specific ATP-dependent system, which uses Ub as a tag to label proteins committed to be hydrolyzed by a specific 26 S protease. PGP-9.5 is another important component of the Ub system, i.e. a neuron-specific carboxyl-terminal hydrolase, which recycles Ub from Ub-polypeptide complexes. We have investigated the expression of Ub and PGP-9.5 in rat hippocampal neurons in an early phase of reperfusion in a model of transient global brain ischemia/hypoxia (bilateral occlusion of common carotid arteries for 10 min accompanied by mild hypoxia-15% O2-for 20 min), by means of immunohistochemical methods using light and electron microscopy. The intensity of Ub and PGP-9.5 immunoreactivity was evaluated by image analysis. We have detected a marked increase of Ub immunoreactivity (UIR) in neurons of CA1, CA2, CA3, CA4, and dentate gyrus subfields 1 hr after ischemia/hypoxia (but not after hypoxia only), statistically significant as confirmed by image analysis. Such increase in immunoreactivity in ischemic/hypoxic rats was localized essentially in the nuclei of hippocampal neurons. There were no changes in PGP-9.5 immunoreactivity. The data suggest that in the present model of rat brain ischemia/hypoxia Ub is involved in the neuronal stress response.

Low environmental radiation background impairs biological defence of the yeast Saccharomyces cerevisiae to chemical radiomimetic agents.

Background radiation is likely to constitute one of the factors involved in biological evolution since radiations are able to affect biological processes. Therefore, it is possible to hypothesize that organisms are adapted to environmental background radiation and that this adaptation could increase their ability to respond to the harmful effects of ionizing radiations. In fact, adaptive responses to alkylating agents and to low doses of ionizing radiation have been found in many organisms. In order to test for effects of adaptation, cell susceptibility to treatments with high doses of radiomimetic chemical agents has been studied by growing them in a reduced environmental radiation background. The experiment has been performed by culturing yeast cells (Saccharomyces cerevisiae D7) in parallel in a standard background environment and in the underground Gran Sasso National Laboratory, with reduced environmental background radiation. After a conditioning period, yeast cells were exposed to recombinogenic doses of methyl methanesulfonate. The yeast cells grown in the Gran Sasso Laboratory showed a higher frequency of radiomimetic induced recombination as compared to those grown in the standard environment. This suggests that environmental radiation may act as a conditioning agent.

Characterization of acetylcholinesterase secretion in neuronal cultures and regulation by high K+ and soluble factors from target cells.

We have observed that cultured neurons from chick spinal cord and the neuroblastoma hybrid line 108CC15 released lower amounts of acetylcholinesterase (AChE) when compared with the parental line, N18TG2. AChE activity extracted by hypotonic buffer, which can be regarded as the source of the released enzyme, was considerably higher in the parental than in the hybrid 108CC15 (respectively, approximately 80% and approximately 40% of cellular activity). On the other hand, evaluation of ectocellular, with respect to total, AChE activity showed that in N18TG2 cells only 7% of AChE was localized on the plasmalemma, whereas in the hybrid line the percentage of ectocellular activity was 3.7 times higher than in the parental line. We have also examined the effect of cytochalasin B and nocodazole. In the N18TG2 line, the former did not affect AChE release, which was significantly reduced by the latter. High K+ level in the culture medium, of both N18TG2 and hybrid 108CC15 cultures, induced an increase in AChE secretion; Ca2+ presence was required for high K(+)-induced release. Muscle extracts increased AChE secretion in both the hybrid 108CC15 and the spinal cord neurons. The present data suggest that AChE secretion during neuronal development is modulated by depolarizing stimuli and by soluble factors produced by target cells and may be involved in the control of neuronal differentiation.

Characterization of 3':5' cyclic nucleotide phosphodiesterase activities of mouse neuroblastoma N18TG2 cells.

Characterization of 'low Km' 3':5' cyclic nucleotide phosphodiesterase activities (PDE) expressed in mouse N18TG2 neuroblastoma cells is reported. At least 3 peaks of activity were isolated by DEAE chromatography, none of which was calcium-calmodulin stimulated and cGMP stimulated or inhibited. A first peak elutes at 200 mM sodium acetate; it specifically hydrolyzes cGMP with a Km of 4.7 microM and shows sensitivity to zaprinast [M&B 22948] (1.8 microM). A second peak eluting at 410 mM sodium acetate hydrolyzes both cyclic nucleotides. A third peak, specific for cAMP hydrolysis, elutes at 580 mM sodium acetate, has a Km of 3.2 microM and is sensitive to RO 20 1724 (7.6 microM) and rolipram (2 microM). Hydrodynamic analysis showed for the first peak a Stokes radius of 5.3 nm with a sedimentation coefficient of 8.1 S, a frictional ratio (f/fo) of 1.41 and a native molecular mass of 182 kDa. The same analysis for peak 3 showed a Stokes radius of 4.1 nm with a sedimentation coefficient of 3.2 S, a frictional ratio of 1.63 and a native molecular mass of 56 kDa. The biochemical features reported for the enzyme eluting in the first peak, and its cGMP-binding activity stimulated by inhibitors of phosphodiesterase activity, demonstrate that it belongs to the PDE V subfamily; on the other hand the cAMP specific enzyme eluting in the third peak can be assigned to the 'RO 20 1724 inhibited' form. The significance of these findings is discussed in relation to the functional characteristics of the N18TG2 cell line.

Acetylcholinesterase distribution in subpopulations of murine thymocyte.

The presence of acetylcholinesterase has been detected in the thymus of several species both biochemically and histochemically. In this study we have investigated the molecular forms and the level of this enzyme in separate compartments of the murine thymus and in different thymocyte subpopulations. Similar levels of acetylcholinesterase activity are present both in thymocytes and in the stromal component. Sucrose density gradient analysis revealed the presence of a single molecular form of about 5 S, presumably a dimeric form. Moreover the results demonstrate a preferential association of AChE with mature thymocyte subsets (Peanut Agglutinin negative and Corticoresistant). This finding correlates with the preferential sensitivity of these cells to cholinergic drugs and supports the hypothesis that acetylcholinesterase modulates the cholinergic effects on thymocytes.

[A Diskimplant surgical technic. First clinical results]. / Approccio chirurgico al Diskimplant. Primi risultati clinici.

A new implantation system (Diskimplant) is examined and clinical results in 26 patients considered after two years.