Alpha-lipoic acid protects the retina against ischemia-reperfusion.

The aim of this study was to examine whether the antioxidant alpha-lipoic acid protects retinal neurons from ischemia-reperfusion injury. Rats were injected intraperitoneally with either vehicle or alpha-lipoic acid (100 mg/kg) once daily for 11 days. On the third day, ischemia was delivered to the rat retina by raising the intraocular pressure above systolic blood pressure for 45 min. The electroretinogram was measured prior to ischemia and 5 days after reperfusion. Rats were killed 5 or 8 days after reperfusion and the retinas were processed for immunohistochemistry and for determination of mRNA levels by RT-PCR. Ischemia-reperfusion caused a significant reduction of the a- and b-wave amplitudes of the electroretinogram, a decrease in nitric oxide synthase and Thy-1 immunoreactivities, a decrease of retinal ganglion cell-specific mRNAs and an increase in bFGF and CNTF mRNA levels. All of these changes were clearly counteracted by alpha-lipoic acid. Moreover, in mixed rat retinal cultures, alpha-lipoic acid partially counteracted the loss of GABA-immunoreactive neurons induced by anoxia. The results of the study demonstrate that alpha-lipoic acid provides protection to the retina as a whole, and to ganglion cells in particular, from ischemia-reperfusion injuries. alpha-Lipoic acid also displayed negligible affinity for voltage-dependent sodium and calcium channels.

Betaxolol, a beta(1)-adrenoceptor antagonist, reduces Na(+) influx into cortical synaptosomes by direct interaction with Na(+) channels: comparison with other beta-adrenoceptor antagonists.

Betaxolol, a beta(1)-adrenoceptor antagonist used for the treatment of glaucoma, is known to be neuroprotective in paradigms of ischaemia/excitotoxicity. In this study, we examined whether betaxolol and other beta-adrenoceptor antagonists interact directly with neurotoxin binding to sites 1 and 2 of the voltage-sensitive sodium channel (Na(+) channel) in rat cerebrocortical synaptosomes. Betaxolol inhibited specific [(3)H]-batrachotoxinin-A 20-alpha-benzoate ([(3)H]-BTX-B) binding to neurotoxin site 2 in a concentration-dependent manner with an IC(50) value of 9.8 microM. Comparison of all the beta-adrenoceptor antagonists tested revealed a potency order of propranolol>betaxolol approximately levobetaxolol>levobunolol approximately carteolol>/=timolol>atenolol. None of the drugs caused a significant inhibition of [(3)H]-saxitoxin binding to neurotoxin receptor site 1, even at concentrations as high as 250 microM. Saturation experiments showed that betaxolol increased the K(D) of [(3)H]-BTX-B binding but had no effect on the B(max). The association kinetics of [(3)H]-BTX-B were unaffected by betaxolol, but the drug significantly accelerated the dissociation rate of the radioligand. These findings argue for a competitive, indirect, allosteric mode of inhibition of [(3)H]-BTX-B binding by betaxolol. Betaxolol inhibited veratridine-stimulated Na(+) influx in rat cortical synaptosomes with an IC(50) value of 28. 3 microM. Carteolol, levobunolol, timolol and atenolol were significantly less effective than betaxolol at reducing veratridine-evoked Na(+) influx. The ability of betaxolol to interact with neurotoxin site 2 of the Na(+) channel and inhibit Na(+) influx may have a role in its neuroprotective action in paradigms of excitotoxicity/ischaemia and in its therapeutic effect in glaucoma.

Flupirtine ameliorates ischaemic-like death of rat retinal ganglion cells by preventing calcium influx.

The effect of flupirtine on the loss of retinal ganglion cells following transient elevation of intraocular pressure (experimental ischaemia) or NMDA-induced excitotoxicity was studied. Ischaemia (60 min) or intravitreal injection of NMDA (20 nmol) caused a decrease in Thy-1 mRNA and Thy-1 immunoreactivity which are associated with ganglion cells. Administration of flupirtine counteracted these changes. Moreover, flupirtine dose-dependently inhibited NMDA-induced 45Ca(2+) influx into cultured cortical neurones and retinal pieces in vitro with maximal inhibition being observed at 200 microM. A similar concentration of flupirtine failed to inhibit kainate-stimulated calcium influx into cultured cortical neurones. In addition, flupirtine had no significant effect on [3H]nitrendipine or [3H]diltiazem binding to cortical membranes. The present studies are consistent with previous findings which suggested flupirtine to act as a NMDA antagonist by a mechanism that still remains to be clarified.

An investigation into the potential mechanisms underlying the neuroprotective effect of clonidine in the retina.

alpha(2)-adrenoceptor agonists, such as clonidine, attenuate hypoxia-induced damage to brain and retinal neurones by a mechanism of action which likely involves stimulation of alpha(2)-adrenoceptors. In addition, the neuroprotective effect of alpha(2)-adrenoceptor agonists in the retina may involve stimulation of bFGF production. The purpose of this study was to examine more thoroughly the neuroprotective properties of clonidine. In particular, studies were designed to ascertain whether clonidine acts as a free radical scavenger. It is thought that betaxolol, a beta(1)-adrenoceptor antagonist, acts as a neuroprotective agent by interacting with sodium and L-type calcium channels to reduce the influx of these ions into stressed neurones. Studies were therefore undertaken to determine whether clonidine has similar properties. In addition, studies were undertaken to determine whether i.p. injections of clonidine or betaxolol affect retinal bFGF mRNA levels. In vitro data were generally in agreement that clonidine and bFGF counteracted the effect of NMDA as would occur in hypoxia. No evidence could be found that clonidine interacts with sodium or L-type calcium channels, reduces calcium influx into neurones or acts as a free radical scavenger at concentrations below 100 microM. Moreover, i.p. injection of clonidine, but not betaxolol, elevated bFGF mRNA levels in the retina. The conclusion from this study is that the neuroprotective properties of alpha(2)-adrenoceptor agonists, like clonidine, are very different from betaxolol. The fact that both betaxolol and clonidine blunt hypoxia-induced death to retinal ganglion cells suggests that combining the two drugs may be a way forward to producing more effective neuroprotection.

Blockade of voltage-sensitive Na(+) channels by the 5-HT(1A) receptor agonist 8-OH-DPAT: possible significance for neuroprotection.

The present study was undertaken to determine whether 5-hydroxytryptamine(1A) (5-HT(1A)) receptor agonists interact with voltage-sensitive Na(+) or N- and P/Q-type Ca(2+) channels to reduce the influx of Na(+) and/or Ca(2+). The 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) inhibited both [3H]batrachotoxinin binding to neurotoxin site 2 of the Na(+) channel in rat cortical membranes (IC(50)=5.1 microM) and veratridine-stimulated Na(+) influx into rat synaptosomes (EC(50)=20. 8 microM). The 5-HT(1A) receptor agonist flesinoxan and the 5-HT(1A) receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide (WAY-100635) also displaced [3H]batrachotoxinin binding with similar affinities to 8-OH-DPAT, but were much less effective in reducing veratridine-stimulated Na(+) influx. All three serotonergic agents also increased [3H]saxitoxin binding to neurotoxin site 1 of the Na(+) channel. In contrast, none of these agents interacted with radioligand binding to N- or P/Q-type Ca(2+) channels. These data show that 8-OH-DPAT directly interacts with voltage-sensitive Na(+) channels to reduce Na(+) influx so providing an additional mechanism to explain how it functions as a neuroprotectant.

Betaxolol, a beta1-adrenoceptor antagonist, has an affinity for L-type Ca2+ channels.

The effect of betaxolol on the specific binding of [3H]diltiazem and [3H]nitrendipine to rat cortical membranes was examined. Betaxolol inhibited specific [3H]diltiazem and [3H]nitrendipine binding with IC50 values of 19.7 and 46.3 microM, respectively. The effect of betaxolol on L-type Ca2+ channels showed little stereospecificity, since similar inhibitions of radioligand binding were observed with both racemic betaxolol and L-betaxolol. The dissociation kinetics of [3H]diltiazem were unaffected by 30 microM betaxolol, whereas it increased the [3H]nitrendipine dissociation rate, thus suggesting that betaxolol directly interacts with the benzothiazepine binding site and allosterically modulates the dihydropyridine binding site. Carteolol, propranolol and timolol were also found to inhibit both specific [3H]diltiazem and [3H]nitrendipine binding to rat cortical membranes, but with less potency than betaxolol. The ability of betaxolol to interact with L-type Ca2+ channels may have a role in its therapeutic effects in the management of systemic hypertension and in reducing neuronal death as occurring in glaucoma.

A long-lasting hypotensive effect of topical diltiazem on the intraocular pressure in conscious rabbits.

The effect of calcium channel blockers on intraocular pressure and aqueous humor dynamics remains still controversial, although preliminary evidence suggests that these drugs may be beneficial in the management of ocular hypertension and low-tension glaucoma. Having previously reported the ocular hypotensive effect of topical nifedipine and verapamil in albino rabbits, the original aim of the present work was to evaluate the effect of topical diltiazem on aqueous humor dynamics in this species. Intraocular pressure was measured with a manual applanation tonometer. The experiments examining the ocular actions of diltiazem were carried out in two stages. In the first one, short term effects of topical diltiazem on intraocular pressure were studied in groups of 13 albino rabbits receiving 8 different doses of the drug in order to obtain a dose-response curve. Tonographies were performed in 13 anaesthetized animals before and 90 min after drug instillation. In a second phase, the persistence of the effect of diltiazem on intraocular pressure was examined in 6 groups of 10 rabbits each receiving three different doses of the drug. Topical diltiazem was found to lower intraocular pressure in a dose-related fashion. The maximum response to diltiazem was greater and the ED50 lower than those previously reported for nifedipine and verapamil. In the tonographic study, diltiazem was shown to reduce the facility of aqueous humor outflow and inflow. Diltiazem exhibited a long lasting effect on intraocular pressure that was again dose-related. Depending on the dose administered, the calculated time necessary for the peak effect to be halved ranged from 0.6 to 7.0 days. Due to the intensity and the persistence of its intraocular pressure-lowering effect, diltiazem shows great potential for the treatment of glaucoma, since a daily or less frequent administration may be enough to control ocular hypertension.

A complex interaction between topical verapamil and timolol on intraocular pressure in conscious rabbits.

Calcium channel blockers have complex actions on aqueous humour dynamics that seem to depend on the route of drug administration. When applied topically, verapamil and nifedipine effectively lower intraocular pressure. However, these drugs also produce a slight reduction in aqueous humor outflow through the trabecular meshwork whereby they could modify the effect of other drugs on intraocular pressure. As calcium channel blockers could be effective in the management of ocular hypertension and low-tension glaucoma, the aim of the present work was to assess the interaction between verapamil and timolol when both drugs are topically applied to the eye of albino rabbits. Intraocular pressure was measured with a manual applanation tonometer. The effects of 5-6 different doses of each drug alone and the effects of five mixtures of both drugs at fixed concentration ratios (timolol: verapamil 4:1, 2:1, 1:1, 1:2 and 1:4) were evaluated. After measuring baseline intraocular pressure, one 50 microliters drop of the different solutions was instilled in the left eye. Measures of the intraocular pressure were repeated at intervals of 30 min until the maximal effect was reached. Each set of experiments was carried out in a group of 9-11 rabbits. Dose-response curves were fitted with a nonlinear regression microcomputer programme. The median effect plot was constructed as proposed by Chou and Talalay (1981, 1983, 1984). In order to analyse the nature of the interaction between both drugs, the observed effect was compared with the theoretically expected one and the combination indices, that relate the doses of verapamil and timolol present in the mixtures with the doses of both drugs separately which are equieffective with the combination, were calculated. The effects of verapamil and timolol followed the principle of the mass action law when administered alone. Nevertheless, no adequate dose-response relationship was obtained when the mixtures of both drugs were applied. In general, the observed effects were lower than the expected ones. Combination indices also indicate the presence of antagonism, except for the lowest concentrations of mixtures where verapamil predominates. In this case, combination indices suggest summation of effects or synergy. Both drugs seem to decrease intraocular pressure by reducing aqueous humor secretion through different mechanisms. However, due to the reduction of aqueous humor outflow caused by verapamil, the ocular hypotensive effect of timolol may be antagonized when high doses of verapamil are administered.

Flesinoxan, a 5-HT1A receptor agonist/alpha 1-adrenoceptor antagonist, lowers intraocular pressure in NZW rabbits.

PURPOSE: alpha1-Adrenoceptor antagonists and 5-HT1A receptor agonists reduce intraocular pressure (IOP) in the rabbit. The aims of this study were firstly, to determine the IOP-lowering effects of flesinoxan and selected other hybrid 5-HT1A receptor agonists/alpha1-adrenoceptor antagonists, and secondly, to investigate the mechanism of action of the IOP response to flesinoxan. METHODS: IOP and total outflow facility were measured in rabbits after administration of hybrid drugs. Inositol phosphates accumulation assays were performed using standard methodologies. RESULTS: Topical unilateral instillation of the drugs caused dose-related reductions of IOP. Comparison of the compounds tested revealed a potency order of WB 4101 > flesinoxan > 5-methyl-urapidil > or = BMY7378 > urapidil. WB-4101 caused a small increase in total outflow facility whereas flesinoxan had no effect. Measurement of the IC50 values for inhibition of phenylephrine-stimulated inositol phosphates accumulation in rabbit iris-ciliary body revealed a potency order of WB 4101 > 5-methyl-urapidil > flesinoxan > BMY 7378 = urapidil. Topical flesinoxan was ineffective in reversing phenylephrine-induced mydriasis, yet, pretreatment with the 5-HT1A receptor antagonists MDL 73005EF and pindolol only partially blocked the hypotensive effect of topical flesinoxan. CONCLUSIONS: The present studies indicate the potent and efficacious IOP-lowering capabilities of flesinoxan and certain other ligands with affinity for 5-HT1A receptors/alpha1-adrenoceptors. The exact mechanisms by which these drugs lower IOP in the rabbit are complex but our results indicate that flesinoxan likely reduces aqueous secretion.

Aqueous humor dynamics in alpha-chymotrypsin-induced ocular hypertensive rabbits.

Aqueous humor dynamics were studied in alpha-chymotrypsin-induced ocular hypertensive rabbits either by tonographic or two-level constant pressure perfusion techniques. A significant correlation was obtained between the values of outflow facility in alpha-chymotrypsin-induced ocular hypertensive rabbits as determined by tonography and constant pressure perfusion. The mean value of tonographic outflow facility in ocular hypertensive rabbits was not statistically different from that found in ocular normotensive rabbits. On the contrary, the estimated rate of aqueous inflow in ocular hypertensive rabbits was about 1.5-fold higher than that of ocular normotensive ones. While topical timolol lowered intraocular pressure and aqueous humor inflow in ocular hypertensive rabbits, pilocarpine did not produce any significant effect. Aqueous humor protein was significantly increased in ocular hypertensive eyes. The results of this study show that accurate measurements of outflow facility can be obtained in alpha-chymotrypsin-induced ocular hypertensive rabbits by tonographic technique. Our data suggest that the long-term ocular hypertension induced by alpha-chymotrypsin in albino rabbits may be secondary to an increase in the rate of aqueous humor inflow, likely produced by a breakdown of the blood-aqueous barrier. This finding strongly conflicts with the hypothesis of trabecular blockage as the cause of alpha-chymotrypsin-induced ocular hypertension in this species.

Topical verapamil lowers outflow facility in the rabbit eye.

Results of studies examining the mechanism of the ocular hypotensive effect of topical calcium channel blockers are controversial. Whereas evidence obtained in perfused human eyes indicates that these drugs lower intraocular pressure by increasing the aqueous humor outflow, tonographic studies in rabbits have revealed that they reduce both the aqueous humor outflow and inflow. In order to clarify such a discrepancy, the aim of this study was to assess whether the effect of topical verapamil on the facility of aqueous humor outflow in the rabbit eye was dose-related. Total outflow facility was determined by two-level constant pressure perfusion in anesthetized rabbits. The effect of 5 different concentrations on aqueous humor outflow at 60 minutes postdrug was studied in groups of 10 rabbits each. Baseline outflow facility was also determined in a group of 15 rabbits. In order to check the reliability of the method for detecting drug-induced changes in aqueous outflow, the effect of pilocarpine was also tested. Topical verapamil was shown to lower outflow facility in the rabbit eye in a dose-related fashion. On the contrary, topical pilocarpine was found to significantly increase outflow facility. Our data indicate that topical verapamil reduces outflow facility in the rabbit eye.

Betamethasone-induced ocular hypertension in rabbits.

The effect of subconjunctivally injected betamethasone on intraocular pressure (IOP) was studied in 85 albino New Zealand rabbits. IOP was measured with a Mentor Model 30 classic pneumatonograph that was manometrically calibrated to the rabbit eye. Ocular hypertension was induced by weekly subconjunctival injections of a betamethasone suspension into the left eye. In one experiment, 70 rabbits were given betamethasone for 4 weeks, while a second group of 10 rabbits received betamethasone for 11 weeks. The short-term effects of subconjunctival injections of betamethasone on IOP were also recorded in a third group of 5 rabbits. Weekly injections over 4 weeks resulted in an increase in IOP in the treated eye, which was prolonged to 11 weeks by repeated weekly injections. A sustained increase in IOP was observed in the treated eye for a period of 7 weeks. During the early hours after betamethasone injection, a transient decrease in IOP was registered in both eyes. The results show that weekly subconjunctival injections of betamethasone cause a predictable increase in IOP in the treated eye which may be suitable for testing the short- and long-term effects of antiglaucoma drugs. Evidence suggesting that endogenous glucocorticoids may play a role in the development of ocular hypertension in humans strengthens the potential value of this glaucoma model.

The effect of topical dihydroergocristine on the intraocular pressure in alpha-chymotrypsin-induced ocular hypertensive rabbits.

Having previously reported that topical dihydroergocristine dose-dependently reduces intraocular pressure in ocular normotensive rabbits with a maximum response and potency higher than those of timolol and pilocarpine, the aim of the present work was to assess the effect of this drug in alpha-chymotrypsin-induced ocular hypertensive rabbits. Intraocular pressure was measured with a pneumatonometer. The experiments examining the effects of dihydroergocristine on intraocular pressure were conducted in 10 albino rabbits in which ocular hypertension was induced by intracameral injection of alpha-chymotrypsin. Intraocular pressure responses to drug vehicle and 5 different doses of topical dihydroergocristine were studied in order to obtain a dose-response curve. Tonographies were also performed in ocular hypertensive rabbits 2 h after vehicle and dihydroergocristine instillation to ascertain the actions of this drug on aqueous humor dynamics. Topical dihydroergocristine was found to lower intraocular pressure in alpha-chymotrypsin-induced ocular hypertensive rabbits in a dose-related manner, with the ED50 of the concentration-response curve very similar to that previously obtained in ocular normotensive rabbits. Data from tonographic studies indicate that dihydroergocristine reduces intraocular pressure in this animal model for glaucoma by decreasing the aqueous humor inflow. Our findings suggest that topical dihydroergocristine may be useful in the treatment of ocular hypertension.

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae.

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.

Voltage-dependent calcium channels in the rat retina: involvement in NMDA-stimulated influx of calcium.

Rises in intracellular Ca2+ induced by activation of glutamate receptors are of ultimate importance for neuronal excitability and pathophysiological processes. In the present study, we aimed to elucidate the types of voltage-dependent Ca2+ channels involved in the NMDA-stimulated influx of Ca2+ into the isolated rat retina by using selective blockers. Additionally, the number of binding sites for radioligands labelling L- ([3H]nitrendipine), N- ([125I]omega-conotoxin MVIIA) and P/Q-type ([125I]omega-conotoxin MVIIC) Ca2+ channels was assessed in the rat retina and, for further comparison, in the rat cortex. Incubation of isolated rat retinas with 100 microM NMDA produced a three-fold increase in the influx of 45Ca2+ that was completely blunted by MK-801, a NMDA receptor antagonist, and partially attenuated (approximately 20%) by tetrodotoxin, a Na+ channel blocker. The L-type Ca2+ channel blocker nifedipine reduced NMDA-stimulated Ca2+ influx in a dose-related fashion, with a maximum reduction of approximately 50%. Similar effects were observed with verapamil and diltiazem. Blockers of N- and P/Q-type Ca2+ channels had no significant effect on the influx of Ca2+ evoked by NMDA. Co2+, a non-specific Ca2+ channel blocker, caused an inhibition of NMDA-stimulated Ca2+ influx similar to that of nifedipine. Therefore, of all voltage-dependent Ca2+ channels, L-type channels appear to make the greatest contribution (up to 50%) to the NMDA-stimulated influx of Ca2+ into the isolated rat retina. This finding contrasts with evidence obtained in brain neurones supporting a role for L-, N- and P/Q-type channels in NMDA-evoked Ca2+ signals. A comparison of the number of radioligand binding sites associated with L-, N- or P/Q-type Ca2+ channels in the rat cortex and retina revealed that such a difference cannot be ascribed to a distinct expression pattern of these channels in both tissues, although some variations were found. Interestingly, a different affinity of [3H]nitrendipine for L-type Ca2+ channels in the rat retina and cortex was observed which may reflect the expression of different classes of L-type channels in these tissues. The ability of L-type Ca2+ channel blockers to attenuate NMDA-stimulated Ca2+ influx may underlie their neuroprotective effects in the retina.

The effect of topical diltiazem on the intraocular pressure in betamethasone-induced ocular hypertensive rabbits.

The effect of calcium channel blockers (CCBs) on intraocular pressure (IOP) remains still controversial, although some preliminary reports suggest that these drugs may be effective in the management of ocular hypertension and low-tension glaucoma. The aim of the present work was to assess the effect of topical diltiazem on IOP in an animal model for glaucoma, the betamethasone-induced ocular hypertension in rabbits. IOP was measured with a manometrically calibrated applanation pneumatonograph. Ocular hypertension was produced in 120 rabbits by weekly subconjunctival injection of a betamethasone suspension into the left eye. The experiments examining the ocular actions of diltiazem were carried out in two stages. In the first one, the ability of topical diltiazem to prevent the rise in IOP induced by betamethasone was studied. In a second phase, the effect of topical diltiazem on IOP in betamethasone-induced ocular hypertensive rabbits was assessed. Diltiazem was topically applied once daily for 5 days a week into the left eye. The effect of five different concentrations of diltiazem was evaluated to obtain dose-response curves. Topical diltiazem was found to prevent in a dose-related fashion the betamethasone-induced IOP rise as well as to reduce IOP in rabbits made ocular hypertensive by weekly subconjunctival injection of betamethasone. Unilateral topical administration did not produce a clear effect on IOP in the untreated eye. This is the first report describing the ocular hypotensive action CCBs in animal model for glaucoma. These findings are in agreement with preliminary evidence suggesting that CCBs may have a beneficial effect in human ocular hypertension.