José Melena

A hypothesis to explain ganglion cell death caused by vascular insults at the optic nerve head: possible implication for the treatment of glaucoma

Some apparent characteristics of ganglion cell death in glaucoma Glaucoma is a progressive optic neuropathy with characteristic optic disc changes and associated visual field defects.1 2 The pattern and progression of visual field loss due to ganglion cell death varies between glaucoma patients suggesting that there is some variability in the magnitude of the insult responsible for the cell loss. Ganglion cell death with a spatial and temporal distribution typical of “glaucoma” can be experimentally induced in animals.3-5 One way to try and mimic glaucoma in experimental animals is to raise the intraocular pressure (IOP).3-6 It is clear from such studies that ganglion cells do not all die at the same time.4-8Furthermore, the rate of deterioration of ganglion cells is proportional to the magnitude of the insult.9 The reason for the initiation of ganglion cell death in glaucoma is unknown, but a number of explanatory theories have been proposed with the vasogenic theory perhaps the most widely accepted hypothesis.2 10-12 Raised IOP is not the sole factor responsible for glaucomatous retinal damage but an important one of a number that have been implicated (Fig 1).12-17 Only 10% of patients with increased IOP (⩾22 mm Hg) have glaucoma and between one third and one half of patients with glaucoma initially do not have elevated IOP.1 18 19 Furthermore, as many as one sixth of patients with glaucomatous damage do not appear to have elevated IOP.1 It is therefore clear that raised IOP is not synonymous with having glaucoma.1 20 21 Nevertheless, high IOP is arguably the most important risk factor (see Fig 1), and it is clearly associated with ganglion cell death in glaucoma patients. Figure 1 Possible causes of ganglion cell death in glaucoma. It is suggested that activation of …

Ganglion cell death in glaucoma: what do we really know?

Glaucomatous optic neuropathy is a chronic process which progresses over many years. Data derived from clinical observations and from animal experiments suggest that the axons of the optic nerve and the retinal ganglion cell somata do not die at the same time but that death can vary between months and many years.1 2 Glaucoma patients have characteristic fields of visual loss which enlarge as the disease progresses. Thus, glaucomatous optic neuropathy may not be a chronic degeneration of the whole of the optic nerve and ganglion cell somata but rather a series of acute losses of individual, or groups of, ganglion cells. It seems therefore reasonable to assume that when a patient is diagnosed initially as having glaucoma, only some ganglion cells are dead, whereas others may range from being “unhealthy” to being “slightly sick” while others are “perfectly normal”. It seems also reasonable to argue that if a neuroprotectant (a substance which reaches the retina to elicit an effect: Table 1) can be applied at some stage before blindness occurs it could be of benefit to the glaucoma patient by either “slowing down” the death process of neurons that has already been initiated to die or perhaps by preventing the initiation of death signals to perfectly healthy ganglion cells. This argument can be made despite the lack of success in the use of neuroprotectants for a variety of other diseases (stroke, epilepsy, Parkinson’s disease, AIDS dementia) where neuronal death is a characteristic.1 3 It is the apparent nature of ganglion cell death in glaucoma, very slow and variable, that makes it more likely that the use of neuroprotectants could be successful. View this table: Table 1 The term neuroprotection in glaucoma implies that the substance used to “protect” the ganglion cells reaches the retina (“direct neuroprotection”) to have an effect. In contrast, …

The β-adrenoceptor antagonists metipranolol and timolol are retinal neuroprotectants: comparison with betaxolol

beta-adrenoceptor antagonists are used clinically to reduce elevated intraocular pressure in glaucoma which is characterised by a loss of retinal ganglion cells. Previous studies have shown that the beta(1)-selective adrenoceptor antagonist, betaxolol, is additionally able to protect retinal neurones in vitro and ganglion cells in vivo from the detrimental effects of either ischemia-reperfusion or from excitotoxicity, after topical application. The neuroprotective effect of betaxolol is thought not to be elicited through an interaction with beta-adrenoceptors, but by its ability to reduce influx of sodium and calcium through voltage-sensitive calcium and sodium channels. In the present study it is shown that the non-selective beta-adrenoceptor antagonists, metipranolol and timolol behave like betaxolol. When topically applied they all attenuate the detrimental effect of ischemia-reperfusion. Protection of the retina was determined by evaluating changes in the electroretinogram and by assessing the loss of mRNA for Thy-1, which is expressed in retinal ganglion cells. In addition, studies conducted on neurones in mixed retinal cultures demonstrated that metipranolol, betaxolol and timolol were all able to partially counteract anoxia-induced cell loss and viability reduction. The influence of timolol was, however, not significant. Within the confines of these investigations, an order of neuroprotective efficacy was delineated for the three beta-adrenoceptor antagonists: betaxolol>metipranolol>timolol. The ability of the beta-adrenoceptor antagonists to attenuate ligand-induced stimulation of calcium and sodium entry into neuronal preparations showed a similar order of effectiveness. In conclusion, the ability to confer neuroprotection to retinal neurones is a common feature of three ophthalmic beta-adrenoceptor antagonists (betaxolol, metipranolol and timolol). A comparison of the effectiveness of the individual compounds in protecting retinal cells in vivo was not possible in these studies. However, in vitro studies show that the capacity of the individual beta-adrenoceptor antagonists to act as neuroprotectants appears to relate to their capacity to attenuate neuronal calcium and sodium influx.

Optic nerve and neuroprotection strategies

AbstractBackground Experimental studies have yielded a wealth of information related to the mechanism of ganglion cell death following injury either to the mylinated ganglion cell axon or to the ganglion cell body. However, no suitable animal models exist where injury can be directed to the optic nerve head region, particularly the unmylinated ganglion cell axons. The process of relating the data from the various animal models to many different types of optic neuropathies in man must, therefore, be cautious.Results Extensive studies on the isolated optic nerve have yielded valuable information on the way white matter is affected by ischaemia and how certain types of compounds can attenuate the process. Moreover, there are now persuasive data on how ganglion cell survival is affected when the ocular blood flow is reduced in various animal models. As a consequence, the molecular mechanisms involved in ganglion cell death are fairly well understood and various pharmacological agents have been shown to blunt the process when delivered before or shortly after the insult.Conclusions A battery of agents now exist that can blunt animal ganglion cell death irrespective of whether the insult was to the ganglion cell body or the mylinated axon. Whether this information can be applied for use in patients remains a matter of debate, and major obstacles need to be overcome before the laboratory studies may be applied clinically. These include the delivery of the pharmacological agents to the site of ganglion cell injury and side effects to the patients. Moreover, it is necessary to establish whether effective neuroprotection is only possible when the drug is administered at a defined time after injury to the ganglion cells. This information is essential in order to pursue the idea that a neuroprotective strategy can be applied to a disease like glaucoma, where ganglion cell death appears to occur at different times during the lifetime of the patient.

Betaxolol, a β1-adrenoceptor antagonist, reduces Na+ influx into cortical synaptosomes by direct interaction with Na+ channels: comparison with other β-adrenoceptor antagonists

Betaxolol, a β1‐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 β‐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 [3H]‐batrachotoxinin‐A 20‐α‐benzoate ([3H]‐BTX‐B) binding to neurotoxin site 2 in a concentration‐dependent manner with an IC50 value of 9.8 μM. Comparison of all the β‐adrenoceptor antagonists tested revealed a potency order of propranolol>betaxolol∼levobetaxolol>levobunolol∼carteololtimolol>atenolol. None of the drugs caused a significant inhibition of [3H]‐saxitoxin binding to neurotoxin receptor site 1, even at concentrations as high as 250 μM. Saturation experiments showed that betaxolol increased the KD of [3H]‐BTX‐B binding but had no effect on the Bmax. The association kinetics of [3H]‐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 [3H]‐BTX‐B binding by betaxolol. Betaxolol inhibited veratridine‐stimulated Na+ influx in rat cortical synaptosomes with an IC50 value of 28.3 μM. 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.

Betaxolol, a β1-adrenoceptor antagonist, has an affinity for L-type Ca2+ channels

Abstract The effect of betaxolol on the specific binding of [ 3 H ]diltiazem and [ 3 H ]nitrendipine to rat cortical membranes was examined. Betaxolol inhibited specific [ 3 H ]diltiazem and [ 3 H ]nitrendipine binding with IC 50 values of 19.7 and 46.3 μM, respectively. The effect of betaxolol on L-type Ca 2+ channels showed little stereospecificity, since similar inhibitions of radioligand binding were observed with both racemic betaxolol and l -betaxolol. The dissociation kinetics of [ 3 H ]diltiazem were unaffected by 30 μM betaxolol, whereas it increased the [ 3 H ]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 [ 3 H ]diltiazem and [ 3 H ]nitrendipine binding to rat cortical membranes, but with less potency than betaxolol. The ability of betaxolol to interact with L-type Ca 2+ channels may have a role in its therapeutic effects in the management of systemic hypertension and in reducing neuronal death as occurring in glaucoma.

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.

Comparative effects of antiglaucoma drugs on voltage-dependent calcium channels

Abstract.Background: Experimental evidence suggests that substances able to interact with voltage-dependent Ca2+ channels (VDCCs) might be beneficial in glaucoma management. It was therefore of significance to show that β-adrenoceptor antagonists used in glaucoma directly interact with L-type VDCCs. In the present study, the affinity of several antiglaucoma drugs (betaxolol, carteolol, levobunolol, timolol, brimonidine, dorzolamide, latanoprost and pilocarpine) for these and other VDCCs was investigated using radioligand binding assays. Experiments were also carried out to assess the effect of antiglaucoma drugs on the NMDA-stimulated Ca2+ influx into isolated rat retinas. Methods: Competition radioligand binding studies to L-, N- and P/Q-type VDCCs were performed in rat cortical homogenates. The effects of antiglaucoma drugs on the NMDA-stimulated influx of 45Ca2+ were studied in isolated rat retinas. Results: Only β-adrenoceptor antagonists significantly interacted with radioligand binding to L-type VDCCs, with betaxolol displaying the highest potency. None of the antiglaucoma drugs tested showed any significant affinity for either N- or P/Q-type VDCCs. Only β-adrenoceptor antagonists attenuated the NMDA-stimulated 45Ca2+ influx into isolated rat retinas, with betaxolol exhibiting at least 10 times higher potency than timolol. Brimonidine, dorzolamide, latanoprost and pilocarpine did not elicit any significant effect on the NMDA-stimulated 45Ca2+ influx. Additional experiments strongly suggested that the effect of betaxolol on the NMDA-stimulated 45Ca2+ resulted from inhibition of L-type VDCCs. Conclusion: Of the antiglaucoma drugs investigated, betaxolol displays the greatest L-type VDCC-blocking activity and this may be of clinical relevance. Such a characteristic could account for some of its described ocular actions.

Expectations in the treatment of retinal diseases: neuroprotection.

Loss of vision leading to blindness is commonly due to photoreceptor or ganglion cell malfunction. No evidence exists to suggest that a lesion associated with other retinal celltypes (Müller cells, horizontal cells, bipolar cells and amacrine cells) can directly lead to blindness. However, in ischaemia for example, impairment of Müller cell and astrocyte functions could lead to the death of retinal neurones including photoreceptors and ganglion cells. A challenge in ophthalmology is therefore to find ways of attenuating photoreceptor or ganglion cell injury as occurs in various diseases. With the increased knowledge of genetics and the finding that gene malfunctions are associated with certain retinal diseases, the use of gene therapy as a method for attenuating loss of photoreceptor function in retinitis pigmentosa and age-related macular degeneration has become a possibility. Great progress has been made in this area in animal studies and it is only a matter of time before gene therapy is tested on humans. Retinal transplantation technology has also advanced over recent years and the results of animal studies have shown some promise, but the visual results from limited human studies have thus far been disappointing. The use of stem cells may provide new opportunities in this area. Many studies have suggested that altered blood flow dynamics in defined ocular blood vessels are a cause for ganglion cell or photoreceptor death in diseases such as glaucoma, ischaemic optic neuropathy, diabetic retinopathy, age-related macular degeneration and retinitis pigmentosa. As a consequence, great efforts are being made to correlate changes in ocular blood flow characteristics with the loss of ganglion cell and/or photoreceptor function in defined diseases so that appropriate treatments can be undertaken. Advancement in this area, however, continues to be slow and controversial. This is because measuring ocular blood dynamics in defined microvessels needs to be performed on human subjects and thus is often indirect, so that interpretation of information is difficult. The use of chemicals or pharmacological agents to attenuate whatever causes the death of photoreceptors or ganglion cells in the various blinding diseases is another procedure worthy of consideration and is the subject of this article. Support for this approach is very strong but is based almost entirely on animal studies. The word “neuroprotection” in ophthalmology has been primarily associated with ganglion cell survival and is hardly used in connection with photoreceptor survival, perhaps because photoreceptors lack certain neurone characteristics (they do not stain positive for neurone-specific proteins like PGP 9.5). The term neuroprotection is applied here to both photoreceptors and ganglion cells.

Blockade of voltage-sensitive Na+ channels by the 5-HT1A 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.