Azithromycin: assessment of intrinsic cytotoxic effects on corneal epithelial cell cultures

Rita Mencucci,1 Domenico E Pellegrini-Giampietro,2 Iacopo Paladini,1 Eleonora Favuzza,1 Ugo Menchini,1 Tania Scartabelli21Department of Specialized Surgical Science – Eye Clinic, 2Department of Health Science Section of Clinical Pharmacology and Oncology, University of Florence, Florence, ItalyPurpose: To compare the cytotoxic effects of preservative-free azithromycin on corneal epithelial cells in vivo with those of preservative-free netilmicin and levofloxacin, and the preservative benzalkonium chloride (BAK).Methods: Rabbit corneal epithelial cells in vitro were incubated for 15 minutes or 6 hours with commercially available ophthalmic preservative-free netilmicin 0.3%, levofloxacin 0.3%, or azithromycin 1.5% preparations or different concentrations of unpreserved azithromycin and different concentrations of BAK. Qualitative analysis was undertaken using phase-contrast optics to examine the morphological aspects of cell cultures and quantitative analysis was undertaken by measuring the release of the cytoplasmic enzyme lactate dehydrogenase into the medium immediately and 24 hours after exposure to drugs. Finally, we observed the wound-healing rate of mechanically injured corneal epithelial cells exposed to each antibiotic ophthalmic preparation for 48 hours.Results: Our results show that both the commercially available unpreserved mono-dose preparation of azithromycin and ophthalmic preparations of azithromycin up to a concentration of 1.5% were virtually devoid of harmful effects under our experimental conditions. This was not significantly different from the results obtained for the other antibiotic preparations (P > 0.05) tested, but was unlike the results obtained for BAK. Azithromycin 1.5% also showed good recovery properties after a mechanical wound test.Conclusion: Under our experimental conditions, unpreserved azithromycin 1.5% showed a much lower toxicity than BAK and did not interfere with the wound-healing process.Keywords: macrolides, toxicity, corneal epithelial cell culture, wound healing, preservative, benzalkonium chlorid

Prokineticins are neuroprotective in models of cerebral ischemia and ischemic tolerance in vitro

Bv8/prokineticin 2 (PK2) is a member of a bioactive family of peptides that regulate multiple functions in the CNS including hyperalgesia, neurogenesis, neuronal survival and inflammation. Recent studies have associated PK2 and prokineticin receptors (PKR) with human diseases, but because their role in neuropathology is still debated we examined whether prokineticins exert a protective or deleterious role in models of cerebral ischemia and ischemic tolerance in vitro. In order to mimic cerebral ischemia, we exposed primary murine cortical cell cultures or rat organotypic hippocampal slices to appropriate periods of oxygen-glucose deprivation (OGD), which leads to neuronal damage 24 h later. Ischemic tolerance was induced by exposing hippocampal slices to a preconditioning subtoxic pharmacological stimulus (3 μM NMDA for 1 h) 24 h before the exposure to OGD. Bv8 (10–100 nM) attenuated OGD injury in cortical cultures and hippocampal slices, and the effect was prevented by the PKR antagonist PC7. The development of OGD tolerance was associated with an increase in the expression of PK2, PKR1 and PKR2 mRNA and proteins and was prevented by addition of the antagonist PC7 into the medium during preconditioning. Both Bv8 at protective concentrations and the NMDA preconditioning stimulus promoted the phosphorylation of ERK1/2 and Akt. These findings indicate that the prokineticin system can be up-regulated by a defensive preconditioning subtoxic NMDA stimulus and that PK2 may act as an endogenous neuroprotective factor through the activation of the ERK1/2 and Akt transduction pathway

Involvement of endocannabinoid signaling in the neuroprotective effects of subtype 1 metabotropic glutamate receptor antagonists in models of cerebral ischemia.

Experimental evidence indicates that metabotropic glutamate (mGlu) receptors of the mGlu1 and mGlu5 subtypes play a differential role in models of cerebral ischemia and that only mGlu1 receptors are implicated in the pathways leading to postischemic neuronal injury. The localization of mGlu1 receptors in GABA-containing interneurons rather than in hippocampal CA1 pyramidal cells that are vulnerable to ischemia has prompted experimental studies that have demonstrated mGlu1 receptor antagonist agents attenuate postischemic injury by enhancing GABA-mediated neurotransmission, thus providing a new viewpoint on the neuroprotective mechanism of these pharmacological agents. In view of the recent discovery of a functional interaction between group I mGlu receptors and the cannabinoid system in the modulation of synaptic transmission, we propose a novel mechanism that predicts that the neuroprotective effects of mGlu1 receptor antagonists on CA1 pyramidal cells are mediated by a mechanism that overcomes the "synaptic circuit break" operated by endocannabinoids on GABAergic transmission

NMDA and DHPG preconditioning induce ischemic tolerance with differential mechanisms in rat organotypic hippocampal slices

Ischemic tolerance is a neuroprotective mechanism by which neurons exposed to a subtoxic insult become resistant to a subsequent lethal stressor. However, the molecular mechanisms underlying the preconditioning response are not yet understood. Because cerebral ischemia and excitotoxicity involve the activation of glutamate receptors, we investigated whether AMPA toxicity was modified following pharmacological preconditioning. For our study, we used organotypic hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to selective injury of the CA1 subregion 24 h later or to 10 \ub5M AMPA for 24 h, which leads to nonselective degeneration of all neuronal populations. We used two models of preconditioning by exposing the slices to subtoxic bouts of either NMDA (3 mM for 60 min) or DHPG (10 mM for 30 min) and 24 h later, to 30 min OGD or AMPA excitotoxicity. After preconditioning, we observed a significant reduction in CA1 damage following our toxic insults. We first performed whole-cell voltage-clamp recordings on control or preconditioned CA1 pyramidal neurons of organotypic slices. 24 h after exposure to NMDA or DHPG preconditioning, AMPA induced-currents were significantly reduced. We then evaluated whether the expression of the GluR1, NR2A and NR2B subunits and of PSD-95 were modified after our preconditioning stimuli, by using western blot analysis performed in postsynaptic densities. Our results show that following NMDA, but not DHPG preconditioning, the expression of GluR1 was significantly reduced. To assess whether the GluR1 reduction induced by NMDA preconditioning was associated with internalization of AMPA receptors, we treated preconditioned slices with the cross-linker bis(sulfosuccinimidyl) suberate and then evaluated the expression levels of GluR1. Western blotting analysis revealed that NMDA, but not DHPG preconditioning, increased the internalization of GluR1. In conclusion, our results show that NMDA and DHPG preconditioning stimuli result in neuroprotection against toxic insults by means of different mechanism

Activation of the histaminergic H3 receptor induces phosphorylation of the Akt/GSK-3 beta pathway in cultured cortical neurons and protects against neurotoxic insults

Stimulation of histamine

Differential mechanisms of tolerance induced by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning

We investigated the molecular events triggered by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning, that lead to neuroprotection against excitotoxic insults (AMPA or oxygen and glucose deprivation) in rat organotypic hippocampal slices, with particular attention on glutamate receptors and on cannabinoid system. We firstly evaluated the protein expression of NMDA and AMPA receptor subunits after preconditioning using western blot analysis performed in post-synaptic densities. We observed that following NMDA, but not DHPG preconditioning, the expression of GluA1 was significantly reduced and this reduction appeared to be associated with the internalization of AMPA receptors. Whole-cell voltage clamp recordings on CA1 pyramidal neurons of organotypic slices show that 24 hr after exposure to NMDA and DHPG preconditioning, AMPA-induced currents were significantly reduced. To clarify the mechanisms induced by DHPG preconditioning, we then investigated the involvement of the endocannabinoid system. Exposure of slices to the CB1 antagonist AM251 prevented the development of tolerance to AMPA toxicity induced by DHPG but not NMDA. Accordingly, the MAG-lipase inhibitor URB602, that increases arachidonoylglycerol (2-AG) content, but not the FAAH inhibitor URB597, that limits the degradation of anandamide, was also able to induce tolerance versus AMPA and OGD toxicity, suggesting that 2-AG is responsible for the DHPG-induced tolerance. In conclusion, preconditioning with NMDA or DHPG promotes differential neuroprotective mechanisms: NMDA by internalization of GluA1-AMPA receptors, DHPG by producing the endocannabinoid 2-AG. (Figure presented.)

Lean body mass is a major determinant of levothyroxine dosage in the treatment of thyroid diseases

Total body weight is usually employed to calculate the amount of l-T(4) to be administered in patients with thyroid diseases. The aim of this study was to evaluate the effect of body composition on l-T(4) requirements. Body composition was assessed by dual energy x-ray absorptiometry in 75 patients on TSH-suppressive l-T(4) therapy after conventional thyroid ablation for differentiated cancer. The mean daily dose of l-T(4) was lower in normal-weight (127.5 +/- 21.3 mug/d) vs. overweight (139.4 +/- 24.5) and obese (151.3 +/- 29.1) subjects. There was a much stronger association between the l-T(4) dosage and lean body mass (P < 0.001, r = 0.667) compared with fat mass (P = 0.023, r = 0.26). Measurement of regional tissue composition showed peripheral lean mass as the best correlate with the dose of l-T(4) (r = 0.679, P < 0.001) whereas no correlation was observed with peripheral fat mass. In conclusion, individual l-T(4) requirements are dependent on lean body mass. Age- and gender-related differences in l-T(4) needs reflect different proportions of lean mass over the total body weight. An estimate of lean mass may be helpful to shorten the time required to attain a stable dose of l-T(4), particularly in subjects with high body mass index values that may be due either to increased muscular mass or to obesity

Lean body mass is a major determinant of levothyroxine dosage in the treatment of thyroid diseases

Total body weight is usually employed to calculate the amount of L-T 4 to be administered in patients with thyroid diseases. The aim of this study was to evaluate the effect off body composition on L-T4 requirements. Body composition was assessed by dual energy x-ray absorptiometry in 75 patients on TSH-suppressive L-T4 therapy after conventional thyroid ablation for differentiated cancer. The mean daily dose of L-T 4 was lower in normal-weight (127.5 ± 21.3 μg/d) vs. overweight (139.4 ± 24.5) and obese (151.3 ± 29.1) subjects. There was a much stronger association between the L-T4 dosage and lean body mass (P &lt; 0.001, r = 0.667) compared with fat mass (P = 0.023, r = 0.26). Measurement of regional tissue composition showed peripheral lean mass as the best correlate with the dose of L-T4 (r = 0.679, P &lt; 0.001) whereas no correlation was observed with peripheral fat mass. In conclusion, individual L-T4 requirements are dependent on lean body mass. Age- and gender-related differences in L-T4 needs reflect different proportions of lean mass over the total body weight. An estimate of lean mass may be helpful to shorten the time required to attain a stable dose of L-T 4, particularly in subjects with high body mass index values that may be due either to increased muscular mass or to obesity