Morphine stimulates angiogenesis through Akt/mTOR/eIF4E activation under serum deprivation or H2 O2 -induced oxidative stress condition.

Morphine is an opioid analgesic drug routinely used to treat pain in several medical conditions including cancer. Increasing evidence has shown that morphine can directly modulate cancer growth via regulating angiogenesis. In this work, we investigated the effect of morphine on angiogenesis under pathological conditions. We showed that morphine, in a concentration typical of that observed in patient's blood, stimulates tumour angiogenesis under serum deprivation and H2 O2 -induced oxidative stress conditions. We found that morphine protected human lung tumour associated-endothelial cell (HLT-EC) against serum deprivation or H2 O2 -induced inhibition of capillary network formation. Furthermore, morphine stimulated other biological functions of HLT-EC under serum deprivation and H2 O2 -induced pathological conditions, such as growth, migration and survival, without affecting HLT-EC adhesion. Interestingly, morphine at the same concentration did not affect lung tumour cell growth and survival, suggesting the specific protective role of morphine at low micromolar concentrations on tumour angiogenesis. Using in vivo Matrigel angiogenesis assay, it was found that morphine stimulated in vivo angiogenesis under H2 O2 -induced pathological condition. The opioid receptor antagonist, naloxone, did not inhibit the protective activity of morphine in in vivo angiogenesis, indicating that the effect was less likely to be mediated by the typical opioid receptors. Mechanism analysis indicated that morphine alleviated serum deprivation and H2 O2 -induced angiogenesis inhibition via reducing oxidative stress and damage, and activating Akt/mTOR/eIF4E signalling. We demonstrate the protective role of morphine on tumour angiogenesis under pathological conditions. Our work suggests that clinical use of morphine may be harmful in patients with angiogenesis-dependent cancers.

Ex vivo model of epilepsy in organotypic slices-a new tool for drug screening.

BACKGROUND: Epilepsy is a prevalent neurological disorder worldwide. It is characterized by an enduring predisposition to generate seizures and its development is accompanied by alterations in many cellular processes. Organotypic slice cultures represent a multicellular environment with the potential to assess biological mechanisms, and they are used as a starting point for refining molecules for in vivo studies. Here, we investigated organotypic slice cultures as a model of epilepsy. METHODS: We assessed, by electrophysiological recordings, the spontaneous activity of organotypic slices maintained under different culture protocols. Moreover, we evaluated, through molecular-based approaches, neurogenesis, neuronal death, gliosis, expression of proinflammatory cytokines, and activation of NLRP3 inflammasome (nucleotide-binding, leucine-rich repeat, pyrin domain) as biomarkers of neuroinflammation. RESULTS: We demonstrated that organotypic slices, maintained under a serum deprivation culture protocol, develop epileptic-like activity. Furthermore, throughout a comparative study with slices that do not depict any epileptiform activity, slices with epileptiform activity were found to display significant differences in terms of inflammation-related features, such as (1) increased neuronal death, with higher incidence in CA1 pyramidal neurons of the hippocampus; (2) activation of astrocytes and microglia, assessed through western blot and immunohistochemistry; (3) upregulation of proinflammatory cytokines, specifically interleukin-1ß (IL-1ß), interleukin-6, and tumor necrosis factor α, revealed by qPCR; and (4) enhanced expression of NLRP3, assessed by western blot, together with increased NLRP3 activation, showed by IL-1ß quantification. CONCLUSIONS: Thus, organotypic slice cultures gradually deprived of serum mimic the epileptic-like activity, as well as the inflammatory events associated with in vivo epilepsy. This system can be considered a new tool to explore the interplay between neuroinflammation and epilepsy and to screen potential drug candidates, within the inflammatory cascades, to reduce/halt epileptogenesis.

Astrocyte truncated tropomyosin receptor kinase B mediates brain-derived neurotrophic factor anti-apoptotic effect leading to neuroprotection.

Astrocytes are glial cells that help maintain brain homeostasis and become reactive in neurodegenerative processes releasing both harmful and beneficial factors. We have demonstrated that brain-derived neurotrophic factor (BDNF) expression is induced by melanocortins in astrocytes but BDNF actions in astrocytes are largely unknown. We hypothesize that BDNF may prevent astrocyte death resulting in neuroprotection. We found that BDNF increased astrocyte viability, preventing apoptosis induced by serum deprivation by decreasing active caspase 3 and p53 expression. The anti-apoptotic action of BDNF was abolished by ANA-12 (a specific TrkB antagonist) and by K252a (a general Trk antagonist). Astrocytes only express the BDNF receptor TrkB-truncated isoform 1, TrkB-T1. BDNF induced ERK, Akt, and Src (a non-receptor tyrosine kinase) activation in astrocytes. Blocking ERK and Akt pathways abolished BDNF protection in serum deprivation-induced cell death. Moreover, BDNF protected astrocytes from death by 3-nitropropionic acid (3-NP), an effect also blocked by ANA-12, K252a, and inhibitors of ERK, calcium, and Src. BDNF reduced reactive oxygen species levels induced in astrocytes by 3-NP and increased xCT expression and glutathione levels. Astrocyte-conditioned medium (ACM) from untreated astrocytes partially protected PC12 neurons, whereas ACM from BDNF-treated astrocytes completely protected PC12 neurons from 3-NP-induced apoptosis. Both ACM from control and BDNF-treated astrocytes markedly reduced reactive oxygen species levels induced by 3-NP in PC12 cells. Our results demonstrate that BDNF protects astrocytes from cell death through TrkB-T1 signaling, exerts an antioxidant action, and induces release of neuroprotective factors from astrocytes. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Anakinra Protects Against Serum Deprivation-Induced Inflammation and Functional Derangement in Islets Isolated From Nonhuman Primates.

We investigated whether serum deprivation induces islet amyloid polypeptide (IAPP) oligomer accumulation and/or a proinflammatory response and, if so, whether the addition of interleukin (IL)-1 receptor antagonist to the culture medium can relieve the proinflammatory response during serum-deprived culture of nonhuman primate (NHP) islets. After culture in medium with and without Ana under serum-deprived culture conditions, IAPP oligomer/amyloid accumulation, in vitro viability, islet function, cytokine secretion, and posttransplantation outcome in streptozotocin-induced diabetic nude mice were determined in islets isolated from heterozygote human IAPP transgenic (hIAPP+/- ) mice and/or NHP islets. Serum deprivation induced accumulation of IAPP oligomer, but not amyloid, in NHP islets. Anakinra (Ana) protected islets from the serum deprivation-induced impairment of in vitro viability and glucose-stimulated insulin secretion and attenuated serum deprivation-induced caspase-1 activation, transcription, and secretion of IL-1ß, IL-6, and tumor necrosis factor-α in hIAPP+/- mice and NHP islets. Supplementation of medium with Ana during serum-deprived culture also improved posttransplantation in vivo outcomes of NHP islets. In conclusion, serum deprivation induced accumulation of IAPP oligomers and proinflammatory responses in cultured isolated islets. Supplementation of the culture medium with Ana attenuated the functional impairment and proinflammatory responses induced by serum deprivation in ex vivo culture of NHP islets.

Protection of seven dibenzocyclooctadiene lignans from Schisandra chinensis against serum and glucose deprivation injury in SH-SY5Y cells.

Dibenzocyclooctadiene lignans, the major active components of fruit of Schisandra chinensis (Turcz.) Baill., have been found to have activities that could prevent prostate and thyroid cancer, hepatotoxicity, oxidative stress-induced cerebral injury, etc. This study was conducted to evaluate the effects of seven dibenzocyclooctadiene lignans of Schisandra chinensis and explore the possible mechanisms in the human neuroblastoma SH-SY5Y cells exposed on serum and glucose deprivation (SGD) injury. The structure-activity relationships were also analyzed. Cell viability and lactate dehydrogenase (LDH) release were determined to evaluate cell injury. Inflammation and apoptosis-related protein levels were detected to elucidate the possible mechanisms. Schisantherin A, schizandrin C, and schizandrol B were found to have stronger protective effects than schizandrin A, schizandrin B, and schisanhenol in SH-SY5Y cells against SGD injury. Moreover, the protective effects of these lignans were possibly exhibited by regulating inflammation and apoptosis-related proteins in SH-SY5Y cells after SGD injury, supporting their beneficial effects for the prevention of cell injury in the pathogenesis of the central nervous system diseases, including ischemia stroke. The number and position of hydroxyl group and methylenedioxy in these lignans may be required for their effects.

Neuroprotective activity of 2-amino-1,3,4-thiadiazole derivative 4BrABT--an in vitro study.

4BrABT (2-(4-Bromophenylamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole) is a compound known for its interesting in vitro anticancer profile. 4BrABT inhibited proliferation and motility of several cancer cell lines in concentrations which were not toxic to normal cells. A major problem associated with cancer chemotherapy, but also caused by environmental factors such as pesticides, is neurotoxicity. Therefore, the aim of the presented study was an in vitro evaluation of the neuroprotective activity of this compound. 4BrABT activity (1-100 µM) was tested in cultures of mouse neurons, rat astrocytes and rat oligodendrocytes. A possible protective action of the compound in different neurodegenerative models, as serum deprivation (SD), excitotoxicity (presence of 500 µM glutamate in culture medium), as well as cisplatin toxicity (astroglia--50 µM and oligodendroglia--100 µM) was investigated. Cell viability in the tested cultures was assessed with the use of LDH and MTT methods. Moreover, 4BrABT ability to prevent the cisplatin-induced apoptosis in astrocyte and oligodendrocyte cultures was analysed after Hoechst 33342 fluorostaining. The obtained results indicate that 4BrABT was not toxic to neurons, astrocytes and oligodendrocytes. Moreover, a decrease in the neuronal LDH level was observed, which may suggest the ability of 4BrABT to act as a trophic agent. Furthermore, the protective action of the studied compound was shown in neuronal cultures exposed to neurotoxic conditions (presence of glutamate and trophic stress) and in cisplatin-treated astrocytes and oligodendrocytes. The expression of anticancer and neuroprotective activity raises hopes for the potential use of 4BrABT as a safe anticancer drug, or neuroprotective agent in chemotherapy-associated neurotoxicity.

5-HT2A serotonin receptor agonist DOI alleviates cytotoxicity in neuroblastoma cells: role of the ERK pathway.

Disturbances of serotonergic signaling, including the serotonin 2A (5-HT2A) receptor, have been implicated in neuropsychiatric and neurodegenerative disorders. The aim of the present study was to characterize the effect of a 5-HT2A receptor agonist on cytotoxicity in a neuronal cell line and address the involved mechanism. HTR2A mRNA and protein expression in human neuroblastoma SK-N-SH cells was confirmed. Cells were subjected to serum deprivation and cell viability was monitored continuously with xCELLigence. In a dose-response study the 5-HT2A agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) (25 nM to 5 µM) protected against serum deprivation cytotoxicity. The selective 5-HT2A receptor antagonist MDL 11,939, the general protein tyrosine kinase inhibitor genistein, and the extracellular signal-regulated kinase (ERK) pathway MEK inhibitor U0126, all attenuated DOI's protective effect. An antibody array suggested that 1 µM DOI affected phosphorylation of several tyrosine kinases. Western blot further confirmed that DOI transiently increased ERK phosphorylation, indicating its activation. Finally, protective concentrations of DOI increased cellular mitochondrial mass, an effect prevented by pretreatment with U0126. In conclusion, our results suggest that DOI protects SK-N-SH cells against serum deprivation through ERK pathway activation. They imply 5-HT2A receptor modulation as a potential target for neuroprotection.

Apotransferrin protects cortical neurons from hemoglobin toxicity.

The protective effect of iron chelators in experimental models of intracerebral hemorrhage suggests that nonheme iron may contribute to injury to perihematomal cells. Therapy with high affinity iron chelators is limited by their toxicity, which may be due in part to sequestration of metals in an inaccessible complex. Transferrin is unique in chelating iron with very high affinity while delivering it to cells as needed via receptor-mediated endocytosis. However, its efficacy against iron-mediated neuronal injury has never been described, and was therefore evaluated in this study using an established cell culture model of hemoglobin neurotoxicity. At concentrations similar to that of CSF transferrin (50-100 micrograms/ml), both iron-saturated holotransferrin and apotransferrin were nontoxic per se. Overnight exposure to 3 µM purified human hemoglobin in serum-free culture medium resulted in death, as measured by lactate dehydrogenase release assay, of about three-quarters of neurons. Significant increases in culture iron, malondialdehyde, protein carbonyls, ferritin and heme oxygenase-1 were also observed. Holotransferrin had no effect on these parameters, but all were attenuated by 50-100 micrograms/ml apotransferrin. The effect of apotransferrin was very similar to that of deferoxamine at a concentration that provided equivalent iron binding capacity, and was not antagonized by concomitant treatment with holotransferrin. Transferrin receptor-1 expression was localized to neurons and was not altered by hemoglobin or transferrin treatment. These results suggest that apotransferrin may mitigate the neurotoxicity of hemoglobin after intracerebral hemorrhage. Increasing its concentration in perihematomal tissue may be beneficial.

Galanin protects against intracellular amyloid toxicity in human primary neurons.

Galanin and galanin receptors are upregulated in the brain regions associated with Alzheimer's disease (AD). However, the consequence of this overexpression is still unknown, particularly in human neurons. Here, we investigate the possible protective effects of galanin against intracellular amyloid-beta (Abeta)1-42 toxicity, as well as other insults including staurosporine, etoposide, hydrogen peroxide, and serum depletion in cultured human primary neurons. The results show that galanin is protective against intracellular Abeta cytotoxicity and all of the above insults at sub-nanomolar physiological concentrations. The galanin protection may be mediated by galanin receptor 2 and down-regulation of Bax level. The data from the present study provide a potential drug target for therapy or prevention of neurodegenerative diseases, including AD.

Sphingosine kinase 1 localized to the plasma membrane lipid raft microdomain overcomes serum deprivation induced growth inhibition.

Several studies have demonstrated that sphingosine kinase 1 (SphK1) translocates to the plasma membrane (PM) upon its activation and further suggested the plasma membrane lipid raft microdomain (PMLRM) as a target for SphK1 relocalization. To date, however, direct evidence of SphK1 localization to the PMLRM has been lacking. In this report, using multiple biochemical and subcellular fractionation techniques we demonstrate that endogenous SphK1 protein and its substrate, D-erythro-sphingosine, are present within the PMLRM. Additionally, we demonstrate that the PMA stimulation of SphK1 localized to the PMLRM results in production of sphingosine-1-phosphate as well as induction of cell growth under serum deprivation conditions. We further report that Ser225Ala and Thr54Cys mutations, reported to abrogate phosphatidylserine binding, block SphK1 targeting to the PMLRM and SphK1 induced cell growth. Together these findings provide direct evidence that the PMLRM is the major site of action for SphK1 to overcome serum-deprived cell growth inhibition.

PACAP and VIP prevent apoptosis in schwannoma cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are structurally endogenous peptides showing rich profile of biological activities. These peptides bind specific membrane receptors belonging to the superfamily of G protein-coupled receptors, the PAC1 and VPAC type receptors. Although these receptors have been identified in oligodendrocytes progenitors cells, to date the effects of PACAP and VIP in Schwann cells are still unknown. In the present study we investigated the expression of these neuropeptides as well as their receptors in a schwannoma cell line. RT-PCR and western blot analysis demonstrated that both PAC1 and VPAC2 receptors, but also PACAP peptide were expressed. To study the physiological effects mediated by PAC1/VPAC receptors, we evaluated their role in preventing apoptotic cell death induced by serum deprivation. Treatment with 100 nM PACAP38 and 100 nM VIP increased survival of serum-deprived schwannoma cells. Anti-apoptotic effects of these peptides were correlated to changes in BCL2 and BAX gene expression. Our results suggested that both PACAP38 and VIP could act as trophic factors in Schwann cells.

The flavonoid baicalin counteracts ischemic and oxidative insults to retinal cells and lipid peroxidation to brain membranes.

The purpose of the present study was to determine whether the flavonoid, baicalin is effective at blunting the negative influence of ischemia/reperfusion to the rat retina in situ and of various insults to a transformed retinal ganglion cells (RGC-5 cells) in culture. Baicalin was administered intraperitoneally just before and after an ischemic insult to retina of one eye of a rat. Ischemia was delivered by raising the intraocular pressure above the systolic blood pressure for 50min. Seven days after ischemia, retinas were analysed for the localisation of various antigens. Retinal extracts were also analysed for various mRNAs. Moreover, the content of specific proteins was deduced in retinal and optic nerve extracts. Also, RGC-5 cells in culture were given one of three different insults, light (1000lx for 2 days), hydrogen peroxide (200microM H(2)O(2) for 24h) or serum deprivation (48h) where cell survival and reactive oxygen species (ROS) formation was assayed. Moreover, a lipid peroxidation assay was used to compare the antioxidant capacity of baicalin with the flavonoid, epigallocatechin gallate (EGCG). Ischemia/reperfusion to the retina affected the localisation of Thy-1 and choline acetyltransferase (ChAT) and the content of various proteins (optic nerve and retina) and mRNAs (retina). Importantly, baicalin statistically blunted most of the effects induced by ischemia/reperfusion. Only the increase in caspase-8 and caspase-3 mRNAs caused by ischemia/reperfusion were unaffected by baicalin treatment. Baicalin also attenuated significantly the negative insult of light, hydrogen peroxide and serum withdrawal to RGC-5 cells. In the lipid peroxidation studies, baicalin was also found to be equally effective as EGCG to act as an antioxidant. Significantly, the negative insult of serum withdrawal on RGC-5 cell survival was blunted by baicalin but not by EGCG revealing the different properties of the two flavonoids.

Prolonged morphine application modulates Bax and Hsp70 levels in primary rat neurons.

Morphine has been used for pain treatment with a long history. Some data suggest that morphine is toxic to neurons and induces apoptosis, while other evidence shows that morphine could have beneficial effects against cell death. To determine how morphine affects pro-apoptotic protein Bax and molecular chaperone Hsp70, different concentrations of morphine were examined. Our results show that prolonged morphine administration for 5 days at 1microM concentration protects against serum deprivation induced cell death in rat primary neurons. Morphine treatment decreases Bax and Hsp70 levels in cultured rat primary neurons, suggesting morphine may have a protective role in staurosporine and serum deprivation induced cytotoxicity.

Cesium chloride protects cerebellar granule neurons from apoptosis induced by low potassium.

Neuronal apoptosis plays a critical role in the pathogenesis of neurodegenerative disorders, and neuroprotective agents targeting apoptotic signaling could have therapeutic use. Here we report that cesium chloride, an alternative medicine in treating radiological poison and cancer, has neuroprotective actions. Serum and potassium deprivation induced cerebellar granule neurons to undergo apoptosis, which correlated with the activation of caspase-3. Cesium prevented both the activation of caspase-3 and neuronal apoptosis in a dose-dependent manner. Cesium at 8 mM increased the survival of neurons from 45 +/- 3% to 91 +/- 5% of control. Cesium's neuroprotection was not mediated by PI3/Akt or MAPK signaling pathways, since it was unable to activate either Akt or MAPK by phosphorylation. In addition, specific inhibitors of PI3 kinase and MAP kinase did not block cesium's neuroprotective effects. On the other hand, cesium inactivated GSK3beta by phosphorylation of serine-9 and GSK3beta-specific inhibitor SB415286 prevented neuronal apoptosis. These data indicate that cesium's neuroprotection is likely via inactivating GSK3beta. Furthermore, cesium also prevented H(2)O(2)-induced neuronal death (increased the survival of neurons from 72 +/- 4% to 89 +/- 3% of control). Given its relative safety and good penetration of the brain blood barrier, our findings support the potential therapeutic use of cesium in neurodegenerative diseases.

The neuroprotective effect of Activin A and B: implication for neurodegenerative diseases.

Activin is a member of the transforming growth factor-beta superfamily which comprises a growing list of multifunctional proteins that function as modulators of cell proliferation, differentiation, hormone secretion and neuronal survival. This study examined the neuroprotective effect of both Activin A and B in serum withdrawal and oxidative stress apoptotic cellular models and investigated the expression of pro- and anti-apoptotic proteins, which may account for the mechanism of Activin-induced neuroprotection. Here, we report that recombinant Activin A and B are neuroprotective against serum deprivation- and toxin- [either the parkinsonism-inducing neurotoxin, 6-hydroxydopamine (6-OHDA) or the peroxynitrite donor, 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1)] induced neuronal death in human SH-SY5Y neuroblastoma cells. Furthermore, we demonstrate for the first time that transient transfection with Activin betaA or betaB significantly protect SH-SY5Y and rat pheochromocytoma PC12 cells against serum withdrawal-induced apoptosis. This survival effect is mediated by the Bcl-2 family members and involves inhibition of caspase-3 activation; reduction of cleaved poly-ADP ribose polymerase and phosphorylated H2A.X protein levels and elevation of tyrosine hydroxylase expression. These results indicate that both Activin-A and -B share the potential to induce neuroprotective activity and thus may have positive impact on aging and neurodegenerative diseases to retard the accelerated rate of neuronal degeneration.

Hydrogen peroxide attenuates insulin-like growth factor-1 neuroprotective effect, prevented by minocycline.

Oxidative stress-induced neuronal death due to hydrogen peroxide overload plays a critical role in the pathogenesis of numerous neurological diseases. Insulin-like growth factor-1 (IGF-1) is important in maintaining neuronal survival, proliferation, and differentiation in the central nervous system. We now report that sublethal doses of hydrogen peroxide attenuated IGF-1 neuroprotective activity on cultured cerebellar granule neurons under potassium and serum deprivation. Interestingly, this attenuation can be prevented by minocycline, an antibiotic that has been shown to have neuroprotective activity in animal models of neuronal injury. Furthermore, hydrogen peroxide also blocked IGF-1's neuroprotection for cortical neurons deprived of neurotrophic factors (B27), which was prevented by minocycline. Our data suggest that inhibition of IGF-1 signaling by hydrogen peroxide may constitute an additional pathway contributing to its neurotoxicity. More importantly, combining minocycline and IGF-1 could be an effective treatment in neurological diseases associated with both oxidative stress and deficiency of IGF-1.

Efficacy and safety of voriconazole as an additive in Optisol GS: a preservation medium for corneal donor tissue.

PURPOSE: To assess the endothelial toxicity and the microbiological efficacy of voriconazole (100 microg/mL) as an antimicrobial additive to Optisol GS. METHODS: A total of 533 donor rims were studied. One half of each donor rim was placed in standard Optisol GS and the other half rim in Optisol GS fortified with voriconazole (100 microg/mL). All rims were refrigerated for 24 hours at 3 degrees C and placed in thioglycolate broth and incubated at 37 degrees C for 7 days. A pair of donor buttons not used in transplantation was stored for 2 days in each solution and examined for endothelial changes with electron microscopy (EM). A second pair of cornea buttons was examined for toxicity by endothelial staining with 0.3% trypan blue and 0.2% alizarin red. RESULTS: Seven of 533 corneal rim cultures were positive for fungal organisms in the Optisol GS group. No rims were positive for fungal growth in the voriconazole-fortified Optisol GS medium. The difference was statistically significant (P = 0.015; Fisher exact test). There was no difference in the cellular morphology of the button stored in voriconazole fortified Optisol GS compared with Optisol GS using EM. In the bioassay, the percentage of nonviable cells in the voriconazole-fortified medium compared with the control medium was nonsignificant (P < 0.05, Student t test). CONCLUSIONS: Voriconazole seems to be safe as a fortifying agent for cornea storage medium. It significantly reduces the rate of positive fungal rim cultures and shows no signs of endothelial cytotoxicity as viewed by EM and by a bioassay of trypan blue and alizarin red.

Efficacy and safety of moxifloxacin as an additive in Optisol-GS a preservation medium for corneal donor tissue.

PURPOSE: To assess the endothelial toxicity and the microbiological efficacy of moxifloxacin (250 microg/mL) as an additive to Optisol-GS. METHODS: Five hundred nine donor rims were studied. One half of each donor rim was placed in standard Optisol-GS and the other half of the rim in Optisol-GS fortified with moxifloxacin (250 microg/mL). All rims were refrigerated for 24 hours at 3 degrees C and placed in thioglycolate broth and incubated at 37 degrees C for 7 days. One pair of donor buttons not used in transplantation stored in each solution was examined for endothelial changes by using electron microscopy (EM). A second pair of cornea buttons was examined for toxicity by endothelial staining with 0.3% trypan blue and 0.2% alizarin red. All endothelial cells that stained (nonviable cells) and nonstained cells (viable cells) were counted, and the ratio of nonviable cells was calculated. RESULTS: The rate of culture-positive donor rims in the Optisol-GS group was 11.9% (61/509) and in the moxifloxacin-fortified Optisol-GS media was 2.5% (13/509). The difference was statistically significant (P < 0.01; chi test). There was no difference in the cellular morphology of the button stored in moxifloxacin-fortified Optisol-GS compared with Optisol-GS using EM. In the bioassay, the rate of nonviable cells in the moxifloxacin-fortified media compared with the control media was nonsignificant (P > 0.05). CONCLUSION: Moxifloxacin (250 microg/mL) seems to be safe as an additive agent for cornea storage media. It significantly reduces the rate of positive rim cultures and shows no signs of endothelial cytotoxicity as viewed by EM and by a bioassay of trypan blue and alizarin red.

Neuroprotective effects of minocycline against in vitro and in vivo retinal ganglion cell damage.

The purpose of this study was to determine whether minocycline, a semi-synthetic tetracycline derivative, reduces (a) the in vitro neuronal damage occurring after serum deprivation in cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed using E1A virus) and/or (b) the in vivo retinal damage induced by N-methyl-D-aspartate (NMDA) intravitreal injection in mice. In addition, we examined minocycline's putative mechanisms of action against oxidative stress and endoplasmic reticulum (ER) stress. In vitro, retinal damage was induced by 24-h serum deprivation, and cell viability was measured by Hoechst 33342 staining or resazurin reduction assay. In cultures of RGC-5 cells maintained in serum-free medium for up to 24 h, the number of cells undergoing cell death was reduced by minocycline (0.2-20 microM). Serum deprivation resulted in increased oxidative stress, as revealed by an increase in the fluorescence intensity for 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), a reactive oxygen species (ROS) indicator. Minocycline at 2 and 20 microM inhibited this ROS production. However, even at 20 microM minocycline did not inhibit the retinal damage induced by tunicamycin (an ER stress inducer). Furthermore, in mice in vivo minocycline at 90 mg/kg intraperitoneally administered 60 min before an NMDA intravitreal injection reduced the NMDA-induced retinal damage. These findings indicate that minocycline has neuroprotective effects against in vitro and in vivo retinal damage, and that an inhibitory effect on ROS production may contribute to the underlying mechanisms.

Overexpression of torsinA in PC12 cells protects against toxicity.

Childhood-onset dystonia is an autosomal dominant movement disorder associated with a three base pair (GAG) deletion mutation in the DYT1 gene. This gene encodes a novel ATP-binding protein called torsinA, which in the central nervous system is expressed exclusively in neurons. Neither the function of torsinA nor its role in the pathophysiology of DYT1 dystonia is known. In order to better understand the cellular functions of torsinA, we established PC12 cell lines overexpressing wild-type or mutant torsinA and subjected them to various conditions deleterious to cell survival. Treatment of control PC12 cells with an inhibitor of proteasomal activity, an oxidizing agent, or trophic withdrawal, resulted in cell death, whereas PC12 cells that overexpressed torsinA were significantly protected against each of these treatments. Overexpression of mutant torsinA failed to protect cells against trophic withdrawal. These results suggest that torsinA may play a protective role in neurons against a variety of cellular insults.