Mark S. Nash

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.

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, …

Photoreceptors are preferentially affected in the rat retina following permanent occlusion of the carotid arteries.

Carotid artery occlusion (two vessel occlusion; 2-VO) for 3 or 9 months causes a suppression of the electroretinogram. However, after 3 months the retinal morphology appears unaffected judging from the localisation of GABA, ChAT, alpha PKC, Thy-1 and GFAP immunoreactivities. Moreover, no difference in NMDA-R1, opsin or Thy-1 mRNA levels were detected. In contrast, after 9 months 2-VO photoreceptor degeneration occurred as indicated by thinning of the outer nuclear layer and reduced Ret-P1 immunoreactivity. All other immunoreactivities appeared normal. These findings were supported by analysis of retinal mRNA levels. We conclude that the major effect of prolonged 2-VO is photoreceptor degeneration.

NMDA induces BDNF expression in the albino rat retina in vivo.

The effect of an intravitreal injection of NMDA on the expression of brain-derived neurotrophic factor (BDNF) in retinal ganglion cells was investigated in rats. Forty-eight hours after intravitreal injection of NMDA retinal ganglion cell BDNF immunoreactivity was practically obliterated, as was the choline acetyltransferase (ChAT) immunoreactivity associated with a subset of amacrine cells. However, 2h following treatment with NMDA the BDNF immunoreactivity and BDNF mRNA associated with the ganglion cells was enhanced while the amacrine cell ChAT immunoreactivity was clearly reduced and the levels of mRNA coding for rhodopsin and Thy-1 did not change. However, 4h after NMDA injection the increase in BDNF mRNA was now no longer apparent. The results show that synthesis of BDNF is increased in the ganglion cells immediately following an insult by NMDA. It is suggested that this is a natural protective mechanism of rat retinal ganglion cells.

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.

Serotonin-2A Receptor mRNA Expression in Rat Retinal Pigment Epithelial Cells

Previous studies have shown that rat retinal pigment epithelial (RPE) cells in culture express 5-HT2-type serotonin receptors coupled to phospholipase C activity. The presented data confirm this observation where it is shown that serotonin induced increases in radioactive inositol phosphates accumulation in RPE cells pretreated with tritiated inositol. This increase was significantly (p < 0.01) attenuated by 1 µM spiperone, ketanserin, mesulergine and metergoline while the same concentration of spiroxatrine or yohimbine had no effect, suggesting the involvement of 5-HT2A receptors. Using reverse transcriptase-polymerase chain reaction the presence of 5-HT2A receptor mRNA was demonstrated in total RNA isolated from rat RPE cell cultures. Amplification of a 5-HT2A receptor mRNA-derived product was additionally confirmed by Southern blot analysis. The combined data demonstrates the existence of functional 5-HT2A receptors in rat RPE cells.

Agonist-induced effects on cyclic AMP metabolism are affected in pigment epithelial cells of the royal college of surgeons rat ☆

Recent work has demonstrated that stimulation of cAMP production via A2-adenosine receptors is reduced in cultured retinal pigment epithelial cells from the RCS rat. Cultured rat RPE cells are also shown to possess beta 2-adrenergic receptors positively coupled to cAMP production. Isoproterenol and salbutamol both stimulate cAMP levels with half maximal (EC50) values of 0.5 and 0.2 microM, respectively. Isoproterenol action is attenuated most effectively by the beta 2-antagonist, ICI 118551, while the beta 1-antagonist, CGP 20712A, is only partially effective. Isoproterenol-stimulated cAMP production is markedly reduced in the RCS rat RPE when compared to control cultures. In passaged RCS rat RPE cells cAMP stimulation by 10 microM isoproterenol was 6.4% of that by control cultures and in primary cultures it was around 75% of controls. The observed EC50 values were 0.4 and 1.3 microM for passaged control and RCS rat RPE cells, respectively. Melatonin negatively influences cAMP production in the RPE via Gi-proteins. Melatonin attenuated the action of forskolin by 51.1% in control rat RPE but only by 18.6% in the RCS rat RPE. The dose-response curve for melatonin shows an approximate 1000-fold shift in potency in the RCS rat. bFGF also has an inhibitory effect on rat RPE cells. bFGF (50ng/ml) attenuated forskolin-stimulated cAMP levels by 61.9% in control rat RPE but had not effect on the action of forskolin in RCS rat RPE. Serotonin (100 microM) potentiates the forskolin-induced stimulation of cAMP by 140.1%. However, unlike isoproterenol, melatonin and bFGF, the action of serotonin on adenylate cyclase appears normal in the RCS rat RPE. We conclude that the defect in the RCS rat RPE is likely to be due to impaired coupling of the components of the adenylate cyclase system and that this is most probably an abnormal interaction of adenylate cyclase with G-protein alpha-subunits.