G. Chidlow

Neuroprotection in relation to retinal ischemia and relevance to glaucoma.

Management of glaucoma is directed at the control of intraocular pressure (IOP), yet it is recognized now that increased IOP isjust an important risk factor in glaucoma. Therapy that prevents the death of ganglion cells is the main goal of treatment, but an understanding of the causes of ganglion cell death and precisely how it occurs remains speculative. Present information supports the working hypothesis that ganglion cell death may result from a particular form of ischemia. Support for this view comes from the fact that not all types of retinal ischemia lead to the pathologic findings seen in glaucomatous retinas or to cupping in the optic disk area. Moreover, in animal experiments in which ischemia is caused by elevated IOP, a retinal abnormality similar to that seen in true glaucoma is produced, whereas after occlusion of the carotid arteries a different pattern of damage is found. In ischemia, glutamate is released, and this initiates the death of neurons that contain ionotropic glutamate (NMDA) receptors. Elevated glutamate levels exist in the vitreous humor of patients with glaucoma, and NMDA receptors exist on ganglion cells and a subset of amacrine cells. Experimental studies have shown that a variety of agents can be used to prevent the death of retinal neurons (particularly ganglion cells) induced by ischemia. These agents are generally those that block NMDA receptors to prevent the action of the released glutamate or substances that interfere with the subsequent cycle of events that lead to cell death. The major causes of cell death after activation of NMDA receptors are the influx of calcium into cells and the generation of free radicals. Substances that prevent this cascade of events are, therefore, often found to act as neuroprotective agents. For a substance to have a role as a neuroprotective agent in glaucoma, it would ideally be delivered topically to the eye and used repeatedly. It is, therefore, of interest that betaxolol, a beta-blocker presently used to reduce IOP in humans, also has calcium channel-blocking functions. Moreover, experimental studies show that betaxolol is an efficient neuro protective agent against retinal ischemia in animals, when injected directly into the eye or intraperitoneally.

Recharacterization of the RGC-5 Retinal Ganglion Cell Line

PURPOSE The transformed RGC-5 retinal ganglion cell line is used widely in glaucoma research. Increased resistance to glutamate was noted in published literature and led to the recharacterization of the RGC-5 cell line. METHODS Characterization of the RGC-5 cell line was performed by sequencing of a region of the nuclear Thy1 gene and mitochondrial DNA sequencing of a region of the d-loop and tRNA(Phe) gene. Marker expression was examined in undifferentiated cells, and cells differentiated with 50 microg/mL succinyl concanavalin A (S Con A) for 3 days. Glutamate sensitivity was examined in undifferentiated and S Con A differentiated cells by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 24-hours of glutamate treatment. RESULTS RGC-5 cells were found to be of mouse (Mus musculus), not rat (Rattus norvegicus), origin by mitochondrial and nuclear DNA analyses. RGC-5 DNA sequenced in a second laboratory was subsequently found to be of M. musculus origin. Cells stained positively for the neuronal markers beta-tubulin and PGP9.5 and for the microtubule-associated protein tau, but not for known markers of ganglion cells such as neurofilaments or Thy1.2, suggesting that they likely represented a lineage of mouse neuronal precursor cells. Differentiation with S Con A did not increase RGC-5 sensitivity to glutamate excitotoxicity or increase the expression of retinal or ganglion cell marker proteins. CONCLUSIONS Investigators using cells designated as RGC-5 should confirm the species to be of rat origin and retinal-specific marker expression before considering their use as retinal ganglion-like cells.

A hypothesis to suggest that light is a risk factor in glaucoma and the mitochondrial optic neuropathies.

The authors propose that light entering the eye interacts with retinal ganglion cell (RGC) axon mitochondria to generate reactive oxygen intermediates (ROI) and that when these neurons are in an energetically low state, their capacity to remove these damaging molecules is exceeded and their survival is compromised. They suggest that in the initial stages of glaucoma, RGCs exist at a low energy level because of a reduced blood flow at the optic nerve head and that in the mitochondrial optic neuropathies (MONs), this results from a primary, genetic defect in aerobic metabolism. In these states RGCs function at a reduced energy level and incident light on the retina becomes a risk factor. Preliminary laboratory studies support this proposition. Firstly, the authors have shown that light is detrimental to isolated mitochondria in an intensity dependent manner. Secondly, light triggers apoptosis of cultured, transformed RGCs and this effect is exacerbated when the cells are nutritionally deprived. Detailed studies are under way to strengthen the proposed theory. On the basis of this proposal, the authors suggest that patients with optic neuropathies such as glaucoma or at risk of developing a MON may benefit from the use of spectral filters and reducing the intensity of light entering the eye.

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.

Rat retinal ganglion cell loss caused by kainate, NMDA and ischemia correlates with a reduction in mRNA and protein of Thy-1 and neurofilament light

Quantification of retinal ganglion cell (RGC) loss/survival following a defined insult to the retina is a prerequisite in order to allow a comparison to be made between the effectiveness of potential neuroprotective drugs. The purpose of the present study was to extend the characterisation of our previously published semiquantitative RT-PCR assay to assess RGC loss/survival. Comparisons were made between the total mRNA levels of the ganglion cell-specific markers Thy-1 and neurofilament light (NF-L) in the retina at specific times after an intravitreal injection of N-methyl-D-aspartate (NMDA) or kainate or after 45 min of ischemia/reperfusion and also between the levels of NF-L mRNA and protein at various times after NMDA injection. Changes in Thy-1 and NF-L immunoreactivities were also observed. NMDA, kainate and ischemia/reperfusion all caused a reduction in the retinal content of Thy-1 and NF-L mRNAs and immunoreactivities. An excellent correlation was observed between the levels of the two mRNAs after these treatments. After NMDA, loss of NF-L mRNA was shown to precede loss of NF-L protein but total loss of each marker was similar after 7 days. The results of the study demonstrate that injury and subsequent death of RGCs, which occurs after ischemia/reperfusion and after intraocular injection of NMDA or kainate, can be followed by measurement of total retinal levels of Thy-1 and NF-L mRNAs and NF-L protein. The assays provides accurate, practical and complementary methods for assessing the potential benefits of neuroprotective drugs on RGCs which have been injured by a variety of experimental modalities.

α-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 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.

Effectiveness of levobetaxolol and timolol at blunting retinal ischaemia is related to their calcium and sodium blocking activities: relevance to glaucoma

Glaucoma is a chronic optic neuropathy in which retinal ganglion cells die over a number of years. The initiation of the disease and its progression may involve an ischaemic-like insult to the ganglion cell axons caused by an alteration in the quality of blood flow. Thus, to effectively treat glaucoma it may be necessary to counteract the ischaemic-like insult to the region of the optic nerve head. Studies on the isolated optic nerve suggest that substances that reduce the influx of sodium would be particularly effective neuroprotectants. Significantly, of the presently used antiglaucoma substances, only beta-blockers can reduce sodium influx into cells. Moreover, they also reduce the influx of calcium and this would be expected to benefit the survival of insulted neurones. Betaxolol is the most effective antiglaucoma drug at reducing sodium/calcium influx. Our electroretinographic data indicated that topical application of levobetaxolol to rats attenuated the effects of ischaemia/reperfusion injury. Timolol was also effective but to a lesser extent. Based on these data we conclude that beta-blockers may be able to blunt ganglion cell death in glaucoma, and that levobetaxolol may be a more effective neuroprotectant than timolol because of its greater capacity to block sodium and calcium influx.

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.