Evaluation of hyperbranched Poly-L-Lysine uptake and toxicity on inner ear derived cell line as a function of concentration and architecture

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Minocycline attenuates gentamicin induced hair cell loss in neonatal cochlear cultures.

Minocycline, a second-generation tetracycline antibiotic used against gram-negative and gram-positive bacteria, protects against a wide range of neurodegenerative disorders by inhibiting caspases, iNOS and the release of cytochrome c. Since aminoglycoside antibiotics damage sensory hair cells in the inner ear by activating caspase-mediated cell death pathways, we hypothesized that minocycline would protect against gentamicin (GM) ototoxicity. To test this hypothesis, postnatal day 3 (P3) rat, cochlear organotypic cultures were treated with GM alone or in combination with minocycline (10-500 microM). Treatment with GM induced a dose-dependent loss of outer hair cells (OHC) and inner hair cells (IHC). Addition of minocycline to the GM-treated cultures greatly reduced the amount of GM-induced hair cell damage in P3 cochlear cultures. The greatest protection was achieved with 100 microM of minocycline. Application of minocycline alone had no adverse effects on hair cell survival. The advantage of this combination therapy is that minocycline prevents GM-induced hair cell loss while helping to suppress the bacterial infection

Nanotechnology in otology

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MDL 28170 attenuates gentamicin Ototoxicity

Aminoglycosides are highly effective antibiotics; however, their clinical utility is severely limited by their nephrotoxic and ototoxic side-effects. The selective destruction of hair cells in the inner ear by aminoglycoside antibiotics is thought to arise from a carefully orchestrated programme of cell death involving caspases and calcium activated proteases or calpains. To more fully evaluate the role of calpains in aminoglycoside ototoxicity, we applied the cell permeant, selective calpain inhibitor MDL 28170, to gentamicin (GM) treated cochlear organotypic cultures and evaluated the degree of hair cell damage at various drug concentrations. Mean hair cell losses in cochlear cultures treated for 24hours with 250, 500 and 1000 μM of GM were 17, 64 and 81%, respectively. Cochlear cultures treated with 200 μM of MDL 28170 alone for 24hours had no adverse effects on hair cell survival. However, the two highest doses of MDL 28170 (500 and 1000 μM) resulted in disorganization of hair cell rows, stereocilia damage and 30–35% hair cell loss. Addition of MDL 28170 to cochlear cultures treated with 500 and 1000 μM GM enhanced hair cell survival in a dose-dependent manner. Two hundred μM of MDL 28170, which by itself had no adverse effects on hair cells survival, significantly enhanced hair cell survival (30–35%), but failed to provide complete protection against GM ototoxicity. Since MDL 28170 can cross the blood-brain barrier and prevent neurodegeneration, it could conceivably be used in vivo to attenuate aminoglycoside ototoxicity

Evaluation of cell viability and cell toxicity in PC12 and OCk-3 as a function of polycation concentration and architecture

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Uptake and toxicity tests of hyperbranched poly-l-lysine on PC12 and OCk-3 cells

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Protective effects of minocycline and MDL 28170 in gentamicin ototoxicity

Gentamicin side-effects on cochlear structures and function are well known, but not the detailed intracellular molecular mechanisms which lead to aminoglycoside induced ototoxicity. Hair cell death occurs by apoptosis, by the activation of enzymatic cascades known to be involved in the programmed cell death, or by the release of cytochrome-c from the damaged mitochondria. In this paper we have investigated the active role of minocycline, a second-generation tetracycline, and of MDL 28170, a selective calpain inhibitor, in the protection of hair cells from GM damage in in vitro organospecific cultures of the organ of Corti. We used cultures from neonatal (P3) rat cochlea, treated with different dosages of GM, alone, or with the two protector drugs. We have observed a dose-dependent OHC and IHC loss. The GM damage was reduced after treatment with both drugs. These results were also supported by the Disk Diffusion Susceptibility Test (Kirby-Bauer Method), in which we used drug treated organs of Corti. The data suggest that minocycline, previously reported to inhibit the release of cytochrome-c, and MDL 28170, prevent GM induced programmed cell death pathway in cochlear HC

Cisplatin-induced apoptosis in human promyelocytic leukemia cells.

Cis-diamminedichloroplatinum (II) cisplatin (CDDP) is an organometallic compound frequently used in anti-cancer therapy, in particular ovarian, testicular, and head and neck tumors. We found cisplatin was effective against human promyelocytic leukemia cell line HL-60, inhibiting cell cycle progression and inducing time- and concentration- dependent cell death. Presence of nuclear fragmentation, caspase-3 cleavage and annexin V positivity suggests cell death occurred by apoptosis, although DNA internucleosomal fragmentation was not detected. In addition, analysis of malondialdehyde (MDA) production and protein carbonylation indicated that cisplatin increased lipid peroxidation and oxidation of cell proteins. This occurrence was prevented by antioxidants such as N-acetylcysteine (N-aC) and glutathione (GSH), which, consistently, were also able to prevent CDDP-induced cell death. Collectively, these findings indicate that, besides growth inhibition, an increase of oxygen radicals and lipid degradation can account for a significant part of CDDP-induced apoptosis

Cisplatin-induced apoptosis in human promyelocytic leukemia cells.

Cis-diamminedichloroplatinum (II) cisplatin (CDDP) is an organometallic compound frequently used in anti-cancer therapy, in particular ovarian, testicular, and head and neck tumors. We found cisplatin was effective against human promyelocytic leukemia cell line HL-60, inhibiting cell cycle progression and inducing time- and concentration- dependent cell death. Presence of nuclear fragmentation, caspase-3 cleavage and annexin V positivity suggests cell death occurred by apoptosis, although DNA internucleosomal fragmentation was not detected. In addition, analysis of malondialdehyde (MDA) production and protein carbonylation indicated that cisplatin increased lipid peroxidation and oxidation of cell proteins. This occurrence was prevented by antioxidants such as N-acetylcysteine (N-aC) and glutathione (GSH), which, consistently, were also able to prevent CDDP-induced cell death. Collectively, these findings indicate that, besides growth inhibition, an increase of oxygen radicals and lipid degradation can account for a significant part of CDDP-induced apoptosis

Ethanolic extract from Hemidesmus indicus (Linn) displays otoprotectant activities on organotypic cultures without interfering on gentamicin uptake.

The ethanolic extract from Hemidesmus indicus (Linn) (Apocynaceae) (Hie) was studied for its otoprotective effects in ex vivo rat organotypic model of gentamicin (GM) toxicity. In organ of Corti organotypic cultures (OC), GM can induce a fast dose-dependent apoptosis of hair cells (HC), both external and internal. We found that, after coadministration of GM and Hie to organotypic cultures, the extract was able to significantly counteract this toxic effect on HC, at the concentration of 25 and 50microg/ml. Interestingly, at these concentrations the extract was present in the cell medium at a concentration 1.6- and 3.3-fold lower than GM, suggesting its otoprotective activity could not merely due to an aspecific inhibition of GM entry. To support this hypothesis, we evaluated the amount of GM present in organotypic cultures after the coadministration of 1.5mg/ml GM and Hie, and found no significant reduction of GM uptake in the presence of 100microg/ml Hie. These data suggest the otoprotective action of Hie derives from specific inhibition of the apoptotic routine induced by GM treatment