5-Hydroxytryptamine1A N.N. OSBORNE, J.P.M. WOOD, J. MELENA, H.M. CHAO, M.S. NASH, agonists: potential use A.J. BRON, G. CHIDLOW in glaucoma. Evidence from animal studies

Abstract retinal ganglion cells with large somata and large-diameter axons, which correspond to the Various classes of compounds exist to lower magnocellular visual pathway, may show an intraocular pressure (lOP) in the treatment of increased vulnerability, although this has been glaucoma. None of them is ideal since some questioned.3 One pOSSible cause of the death of patients respond better than others and the ganglion cells is mechanical damage and/ or side effects vary between individuals. New ischaemic damage to their axons at the level of classes of compounds need to be introduced to the lamina cribrosa induced by either elevated allow the clinician greater scope for effective intraocular pressure (lOP) or altered blood flow. treatment of all patients. It is now generally Another possible cause may be related to the agreed that the cause of ganglion cell finding that glutamate levels in the vitreous dysfunction in glaucoma is likely to be humour of glaucoma patients are elevated.4 multifactorial and that concentrating solely on This glutamate could have originated from an reducing lOP is inadequate. Irrespective of the injured retina, such as one that had experienced reason for the dysfunction, the future goal an ischaemic-like insult (hypoxia/ anoxia) must be to attenuate cell death. This may be l . An achieved with drugs that interact with caused by reduced blood flow or raised OP components of the retina, and is termed insult of this nature, which leads to the release 'neuroprotection'. Thus, drugs that can both of glutamate, would make retinal neurone-types reduce lOP and act as neuroprotectants would (ganglion cells and a subset of amacrine cells) be ideal for the treatment of glaucoma. In this that contain ionotropic glutamate receptors article we summarise studies on animals particularly vulnerable. Anatomical studies on which show 5-HTIA agonists to retinas of glaucoma patients support the both reduce lOP when topically applied to the opinion that both ganglion cells and certain rabbit eye and blunt the damaging effect to the amacrine cells are particularly affected (see N.N. Osborne rat retina and ganglion cells induced by review by Osborne et aI.5). In experimental l.P.M. Wood glutamate toxicity or ischaemia. Reduction of ischaemia, glutamate is released from the l. Melena 6 7 lOP occurs via stimulation of 5-HT1A receptors retina • and causes destruction of ganglion and H.-M. Chao some amacrine cells.s The released glutamate M.S. Nash associated with the ciliary processes. Al. Bron Neuroprotection of retinal neurones appears to may well accumulate in the vitreous humour. G. Chidlow involve the interaction of 5-HT lA agonists Chronic glaucoma, for which the model is Nuffield Laboratory of with membrane sodium channels and/or primary open-angle glaucoma (POAG), is Ophthalmology 5-HT1A or even possibly 5-HT7 receptors. perhaps best defined as a heterogeneous group University of Oxford Oxford, UK Various 5-HTIA agonists are used in patients of disorders with a distinctive type of optic to treat depression, so classes of these drugs nerve damage and characteristic form of field NN Osborne � have a proven safety profile for use in patients. loss. The major causes of glaucoma are likely to Nuffield Laboratory of The animal studies summarised in this article be hypoxia/anoxia (affected ocular blood Ophthalmology suggest that 5-HT lA agonists need to be lOP. University of Oxford supply to optic nerve head) and/ or raised Walton Street considered as a new class of drugs for the Therefore, the ideal drug for treatment of Oxford OX2 6AW, UK treatment of glaucoma. glaucoma is a substance which, when topically Tel: +44 (0)1865 248996 applied, reduces raised lOP, facilitates ocular Fax: +44 (0)1865 794508 Key words Glaucoma, 5-HydroxytryptaminelA blood flow in the optic nerve head and protects e-mail 9 agonists, Intraocular pressure, Neuroprotection the retina from destruction. Experimental neville. [email protected] studies on animals now give us good reason to Financial support British believe that certain members of two classes of Council for the Prevention In chronic glaucoma the ganglion cells of the substances - l3-blockers (e.g. betaxolol) and of Blindness and the retina appear to die at a slow and variable rate. (5-hydroxytryptamine; 5-HT) 5-HT1A National Eye Research Anatomical and physiological studies of receptor agonists (e.g. 8-hydroxy-2-(di-N­ Centre. l.M. supported by a 1 Marie Curie grant glaucoma in humans and experimental propylamino)tetralin or 8-0H-DPAT and 2 (European Commission) glaucoma induced in monkeys suggest that ) - may fulfil these criteria. Betaxolol

454 Eye (2000) 14, 454-463 © 2000 Royal College of Ophthalmologists Table 1. The concentration of biogenic amines in the human aqueous Table 3. Effects of various antagonists (1 pM) on the 5-HT (1 mM) humour induced accumulation of inositol phosphates (InsPs) in the rabbit iris-ciliary body Amine Concentration (ng/ml) 0.2 % increase in InsPs accumulation Noradrenaline Agent relative to the effect of 5-HT Serotonin 50 5-HT 100 IS 5-HT + 38 ± 8* From Martin et al. 5-HT + 57 + 6* 5-HT + 90 ± 3 is already used to treat glaucoma patients and as a 5-HT + 66 ± 5* consequence our studies on the neuroprotective 5-HT + MDL 72222 80 ± 9 properties of betaxolol are of particular importance (for 5-HT + 80 ± 7 details see Osborne et aI.lO,ll). However, in this article our Data are mean ± SEM value for three or four experiments. A *p < 0.05 by Student's t-test when compared with 5-HT. studies on 5-HT l agonists will be summarised as they represent a new class of compounds for possible use in glaucoma. 5-HTIA agonists, such as and 8-0H-DPAT was originally thought to be a very selective , are presently used in the treatment of 5-HT lA agonist23 but subsequently has been shown to 12 24 depression. 8-0H-DPAT and flesinoxan are two of the have some affinity for the 5-HT7 receptor. 8-0H-DPAT most potent and selective 5-HTIA receptor agonists and when applied topically to the rabbit eye reduces lOP in 25 2 these drugs have proved invaluable in the study of both the light and dark. , 6 The full 5-HT1A agonist 5-HTIA receptor function. (+)8-0H-DPAT is a more effective hypotensive agent than the partial agonist (-)8-0H-DPAT (Fig. 3), and this result when taken with the finding that the effect of Serotonin receptors in the anterior uvea (±)8-0H-DPAT on lOP was blocked by pretreatment Indirect evidence suggests that some serotonergic nerves with , a mixed 5-HTIA antagonist/j3-blocker, but may exist in the iris-ciliary body of rabbits.13 More not by the specific j3-blocker betaxolol, suggests that compelling data show that serotonin is present in the 8-0H-DPAT lowers lOP via activating 5-HT1A receptors human14 and rabbit13 iris-ciliary body as well as the (Fig. 4).26 Recent studies with a number of 5-HTIA aqueous humour, where the concentration of the amine is agonists / cx l-adrenoceptor antagonists (e.g. flesinoxan, greater than that of noradrenaline (Table 1).15,16 Moreover, WB 4101) provide support for the view that 5-HTIA binding (Table 2) and secondary messenger studies (Table agonists reduce lOP. Flesinoxan is not only highly potent 3) on iris-ciliary body tissue from the rabbit suggests that but also a full agonist at 5-HT1A receptors, yet is a 13 17 7 5-HTIA and 5-HT2 receptors are present. , We have relatively weak arblocker? When applied topically to recently shown that mRNAs encoding the 5-HT lA and 5- the rabbit, it causes a dramatic reduction in lOP (Fig. 5). HT7 receptors are associated with the iris-ciliary body Confirmation that the effect involves 5-HTIA receptors complex of the rabbit and that the 5-HTIA receptors are was obtained by use of 5-HTIA antagonists, which primarily associated with the epithelial cell bilayer of the partially nullify the lOP response of the rabbits to ciliary processes (Figs. 1, 2).18 Studies on the isolated flesinoxan (unpublished observations). human iris-dliary body complex also support the Studies with ketanserin also suggest that antagonists of 19 existence of 5-HT1-type receptors and our preliminary 5-HT2 receptors, when applied topically, can lower lOP. experiments indicate that the mRNA which encodes the 5- For example, topically applied ketanserin lowers lOP in the 21 2 29 HTIA receptor is present (unpublished data). rabbit and in man. 8, It should, however, be borne in mind that ketanserin is known to have an affinity for CXl­ adrenoceptors27 and for this reason the effects of ketanserin lOP Effect of serotonergic drugs on on lOP may not be entirely caused by its effect on 5-HT2 Topical application of 5-HT has been reported to both receptors. Additional studies need to be conducted with elevate2D and lower21 lOP in rabbits. In addition, specific 5-HT 2 ligands to clarify the situation. 5-methyl- (a combined 5-HT1A agonist/ 22 cxl-adrenoceptor antagonist) reduces rabbit IOP. Serotonin receptors in the retina

The evidence for serotonin being a neurotransmitter in Table 2. Apparent affinity values (K) of various drugs for eHJ5-HT the mammalian retina (as opposed to the non­ binding to 5-HT1A receptor sites in rabbit iris-ci/iary body membranes mammalian retina) remains a matter of debate because Ki Ligand value (M) there is a lack of clear data showing the existence of 1 DP-5-CT 2.5 X 10- 0 serotonergic neurones?D,31 However, numerous studies MDL 73005EF 3.3 X 10-10 1 have shown serotonin receptors to be associated with 5-CT 4.9 X 10- 0 9 5-HT 2.5 X 10- mammalian retinas. Using radiolabelled serotonin or its 9 8-0H-DPAT 3.6 X 10- analogues, saturable 5-HT binding sites were shown to 4 32 Ketanserin > 1.0 X 10- be associated with the retina; moreover, multiple 5-HT -4 > 1.0 X 10 receptor subclasses were identified in the rabbit retina, 33 34 Data are mean ± SEM value for three or four experiments. including 5-HTI-like and 5-HT2 and 5-HT3.

455 Fig. 1. Agarose gel electrophoresis of PeR-amplified products of RNA from rabbit ciliary processes (lanes 1 and 4), cornea (lanes 2 and 5) and retina (lanes 3 and 6). The single band located at 357 bp seen in lane 1 corresponds to the expected length of the cDNA product produced bythe 5- HT1A receptor primers. The single band located at 464 bp seen in lanes 5 and 7 corresponds to the expected length of the cDNA product produced by the 5-HT 7 receptor primers. No band is detectable in lanes 2 and 3 or lane 6. The 123 bp ladder is shown in between (M).

Secondary messenger studies also showed that serotonin the retina. In the context of glaucoma we therefore have increased cAMp35,36 and inositol phosphate no definitive evidence for ganglion cells containing a production,37,38 demonstrating that retinal 5-HT particular type of serotonin receptor. receptors are coupled functionally to their known effector systems. At the time it was thought that the Neuroprotective action of 5-HT1A agonists serotonergic receptors in the rabbit retina belonged to the 5-HT'A subtype because of the pharmacological profile The direct neuroprotective action of 5-HTIA agonists was of the observed response. However, although early demonstrated by use of confluent rat cortical cultures, studies indicated that 5-HTIA receptors can both which were prepared as previously described.45 The 39 40 stimulate and inhibit adenylate cyclase, it is now cultures in serum-free medium were exposed to widely accepted that 5-HT1A-like stimulations in cAMP glutamate (100 f.lM) either alone or together with a production are mediated not through 5-HTIA receptor 5-HTIA agonist (100 f.lM flesinoxan or 8-0H-DPAT) for activation but via the operationally similar 5-HT7 4 h under normotoxic conditions. In other experiments subtype. Thus the increase in cAMP found in retinal the cultures were subjected to hypoxic conditions, 95% tissue following application of serotonin is most likely N2/5%C02, for 5 hat 37°C with 5-HTIA agonists added due to activation of 5-HT7 receptors, which are before hypoxia where appropriate: reoxygenation was preferentially linked to an increase in cAMP synthesis. achieved by re-placing the cells in normoxic conditions Support for this opinion has come from recent work for 3 h before analyses. Cellular injury (due to 1 which established the presence of 5-HT7 mRNA 8 and L-glutamate or hypoxia/normoxia) was assessed by 5-HT ,like binding sites in the rabbit retina (Figs. 1, 2). measurement of lactate dehydrogenase (LDH) release 45 Although 5-HT1A receptors were not detected in rabbit into the cell culture medium. As shown in Table 4 both retina it could equally not be concluded that they are flesinoxan and 8-0H-DPAT significantly counteracted absent, and indeed, preliminary RT-PCR data indicate the effect of L-glutamate or hypoxia/reperfusion. The that 5-HTIA mRNA is present in human retina. 5-HTIA antagonist/l3-blocker, (5 f.lM), did Neuropharmacological studies suggest that activation not counteract the effect of flesinoxan or 8-0H-DPAT of 5-HTIA' 5-HT2A and 5-HT3 receptors affect visual (Table 4). However, since propranolol had a protective 41 43 processing - and 5-HT2A receptors are associated with effect on its own (Table 4) (probably because of its terminals of the photoreceptors and rod bipolar cells in calcium channel blocking properties46), these the rabbit retina.44 However, as far as the other serotonin experiments could not determine whether the effects of receptors are concerned (5-HTIA' 5-HT3 and 5-HT7 flesinoxan and 8-0H-DPAT are via activation of 5-HT1A receptors), no information exists on their distribution in receptors.

456 Fig. 2. Distribution of 5-HT1A/7 (A, B) and 5-HT1A (E, F) binding sites and 5-HT7 mRNA (c, D) in sections of the rabbit eye as shown by 3 receptor autoradiography and ISHH. (A) [ H]5-carboxamidotryptamine (2 nM). (B) eH]5-carboxamidotryptamine (2 nM) + 5-HT (5 pM). 3 3 3 (C) 5S-labelled 5-HT7 antisense probe. (0) 35S-labelled 5-HT7 sense probe. (E) [ H]WAY 100-635 (3 nM). (F) [ H]WAY 100-635 (3 nM) + 5-HT (5 11.M). Specific 5-HT1A/7 binding sites are observed in the retina and ciliary body (A), while 5-HT1A sites are only apparent in the ciliary body (E). Specific 5-HT7 mRNA hybridisation signals appear to be found in the ciliary body, retina and possibly iris (C). ce, ciliary epithelium; r, retina.

457 4 2

1 ,-, 2 ,-, Q,I Q,I ...... Q,I Q,I 0 e e .. 0 .. = = ·1 = 8 y I I � � Q,I ·2 Q,I ·2 'C � � co ·3 � ·4 � '-'.. '-'.. =- * =- ·4 0 0 ...... ·6 * --0-- (.) 8·0H·DPAT * ·5 -+- pindolol + 8·0H·DPAT -+- (+) 8·0H·DPAT --0-- 8-OH·DPAT

·8 ·6 0 1 2 3 4 5 0 1 2 3 4 5 Time (hours) time (hours)

Fig. 3. Effect of 30 ILl of 0.1% (+)8-0H-DPAT and 0.1% (-)8-0H­ Fig. 4. Effect of topical administration of 30 ILl of 1 % pindolol on the DPAT on lOP in the dark. Contralateraleyes of rabbits received 30 ILl lOP response of normotensive NZW rabbits to 30 ILl of 0.25% 8-0H­ of 0.9% NaC!. Data are represented as mean (:t: SEM) value calculated DPAT. Data are represented as mean (:t: SEM) value calculated by by subtracting the lOP of contralateral eyes from the lOP of treated subtracting the lOP of contralateral eyes from the lOP of treated eyes, eyes, where n = 5. Baseline lOPs varied from 28 to 30 mm Hg. where n = 9. Contralateral eyes of rabbits received 30 ILlof 0.9% NaC!. *p < 0.05 by Student's paired t-test (treated eyes vs contralateral eyes). *p < 0.05 by Student's paired t-test (treated eyes vs contralateral eyes).

22 ,------, period) the electroretinogram (ERG) was recorded from both eyes and the amplitude of the b-wave determined. ,-, The animals were then killed and sections of the retinas CiI 20 == 'stained' for the localisation of choline acetyltransferase e (ChAT) and parvalbumin immunoreactivities. e '-' 18 Ischaemia/reperfusion causes a reduction of the b-wave � (Fig. 6) and changes in the ChAT and parvalbumin fi '" immunoreactivities (Fig. 7). 8-0H-DPAT significantly � 16 =- counteracted the effects of ischaemia/reperfusion so that .... .! the nature of the b-wave and the staining of the ChAT B 14 and parvalbumin appeared more normal (Figs. 6, 7). In 1982 we showed that the antigen Thy-1 is localised ! 48 .S 12 to ganglion cells and subsequent studies have deduced --0-- water * that ischaemia/reperfusion to the rat retina causes -+- flesinoxan destruction of the ganglion cells and loss of the mRNA49 and the antigen50 for Thy-I. By quantifying the mRNA ·60 o 60 120 180 240 300 for Thy-1 in the whole of the retina and relating the time (mins) content to other retinal mRNAs (cyclophilin, rhodopsin) an assay for ganglion cell survival was constructed.49 Fig. 5. Effect of topical administration of 30 ILl of 2% flesinoxan on As shown in Fig. 8, intravitreal injection of N-methyl-D­ the lOP of normotensive NZW rabbits. Data are represented as mean aspartate (NMDA; 20 nmol) to the rat retina causes a :t: SEMfrom 6 rabbits. *p < 0.05, by Student's paired t-test (treated vs contralateral). significant reduction in retinal Thy-1 mRNA level relative to cyclophilin, while this is not the case for rhodopsin mRNA. However, when 8-0H-DPAT To test the general neuroprotective properties of (20 nmol) is co-injected into the vitreous humour (to give 8-0H-DPAT on the rabbit retina in vivo, 8-0H-DPAT was a concentration of 200 fLM in the vitreous humour) at the injected directly twice into the vitreous humour same time as NMDA the reduction in the retinal (concentration in the vitreous humour on each occasion Thy-l mRNA level is significantly attenuated (Fig. 8). 100 fLM) of one eye, once just before ischaemia and then We conclude from such experiments that NMDA­ at the onset of reperfusion. Control animals received induced toxicity to the ganglion cells is attenuated by injections of vehicle in one eye. Ischaemia was induced in 8-0H-DPAT. the injected eye by raising the lOP above the systolic Our most recent studies show that 8-0H-DPAT can blood pressure using a suction-cup procedure47 for act as a retinal neuroprotectant even when applied 60 min. Three days after ischaemia (the reperfusion topically to the eye. It is assumed that the topically

458 Table 4. The effect of 8-0H-DPAT, f/esinoxan, propranolol and MK-801 on the glutamate and hypoxialreperfusion-induced release of lactic dehydrogenase (LDH)

Treatment % of LDH released in comparison with total LDH

Control 10 ::':: 2 (n = 10) Glutamate (50 f.LM) 29 ::':: 5 (n = 10) Glutamate + MK-801 (5 f.LM) 10 ::':: 3 (n = 4) Glutamate + 8-0H-DPAT (100 f.LM) 18 ::':: 4 (n = 4) Glutamate + Flesinoxan (100 f.LM) 17 ::':: 2 (n = 4) Glutamate + Propranolol (5 f.LM) 19 ::':: 4 (n = 4)

Control 12 ::':: 3 (n = 5) H/Ra 23 ::':: 6 (n = 5) H/Ra + MK-801 (5 f.LM) 12 ::':: 4 (n = 3) H/Ra + 8-0H-DPAT (100 f.LM) 14 ::':: 3 (n = 3) H/Ra + Flesinoxan (100 f.LM) 13 ::':: 5 (n = 3) H/R" + Propranolol (5 f.LM) 15 ::':: 4 (n = 3)

The basal (control) release of LDH into the culture medium was 21 ::':: 4mU/ml/mg protein (n = 15). Results are mean::':: SEM. Number (n) of experiments is shown in parentheses. All the drugs significantly (p < 0.05; using one-way ANOVA followed by Dunnett's test) reduced the effect of glutamate or hypoxial reperfusion. Note: MK-801 is an NMDA .

applied drug reaches the retina through the systemic as a free radical scavenger, studies have been conducted system, as this appears to be the case in experiments to determine whether it can act as a 'calcium channel ll where betaxolol was used. In these experiments, the blocker'. Initial experiments were carried out on rat drug (or vehicle) was topically applied (5 j-Ll of 0.5% cortical cultures as described elsewhere.5 3 The cortical solution) 10 min and 5 min before ischaemia, directly cultures in a medium containing 45Ca2+ are exposed to after ischaemia and then daily during reperfusion. The either to NMDA alone or in combination with b-wave of the ERG was also measured at 2 and 5 days 8-0H-DP A T. As shown in Table 5, NMOA stimulates an during reperfusion. Fig. 9 shows that the b-wave of the influx of 45Ca2+ into the neurones and 100 j-LM ERG in the 8-0H-DPAT treated animals is significantly 8-0H-OPAT causes a significant attenuation. Similar less reduced (n = 4) than in those animals which received results have been generated with betaxololY We next vehicle, thus suggesting a 'protection' against ischaemia/ investigated whether 8-0H-OPAT has an affinity for the reperfusion. Moreover, the ChAT immunoreactivity L-type calcium channel using a radioactive binding (5 days follOWing reperfusion) was completely procedure described elsewhere.46 Surprisingly, obliterated in retinas which received ischaemia in all 8-0H-OPAT had very little affinity for the L-type vehicle-treated animals (n = 4) but traces of ChAT calcium channel (Fig. 11). Since betaxolol binds to the immunoreactivity were evident in three of the four animals which received 8-0H-DPAT (Fig. 10).

0.4 - n=6 Neuroprotective mode of action of 8-0H-DP AT ,-, n=S � I n=6 8-0H-DPAT is a potent agonist at 5-HT1A and a partial � T 24 = 0.3 - ** agonist at 5-HT7 receptors, and both receptor-types '-' Q,j � exist in the mammalian retina (see above). Thus the -= neuroprotective effect of 8-0H-DPAT on ischaemia/ .� is. - reperfusion or NMDA-induced effects on the b-wave of 0.2 � n=S .� the ERG, ChAT and parvalbumin immunoreactivities -; Q,j * '" � � -=c and ganglion cell Thy-1 mRNA levels may be mediated \\I � 'a E-< .. .. < � ... 'a via an action on 5-HT1A and/or 5-HT receptors. Studies I - ...... 7 � 0.1 ...... t'� .. . $ .. .$ • on brain tissues have demonstrated that neuroprotection 5 5= 51 52 "S can be induced by activation of 5-HT A receptors. , b .! ] 1 = .!� Q § However, in our studies we could not draw such a ... .� ... .� conclusion because we did not blunt the neuroprotective 0.0 effects of 8-0H-DPAT with propranolol. In addition, Fig. 6. Two groups of animals (n = 5) were used. The right eye of one propranolol is not a very effective 5-HTIA antagonist and group received 10 ILl saline before and after ischaemia; the left eye more selective and specific antagonists need to be tested. received 8-0H-DPAT (final concentration in vitreous humour Laboratory studies show that there are various ways estimated to be 100 ILM) at the same times. The left eye of both groups of attenuating neuronal death caused by ischaemia/ of animals received ischaemia with a suction methodology for 60 min. reperfusion.5 Substances, for example, which prevent an The amplitudes of the b-wave of the electroretinogram was determined 3 days after ischaemia, i.e. following 3 days of reperfusion. It can be unusual rise in calcium and/or free radicals in an seen that administration of 8-0H-DPAT attenuates the reduction in 5 insulted neurone are likely to act as neuroprotectants. the b-wave relative to the control. *p < 0.05 compared with control; Although we have not tested whether 8-0H-DPAT acts **p < 0.05 compared with ischaemia.

459 Fig. 7. (Al) and (Bl) The normal distribution of parvalbumin and choline acetyltransferase (ChAT) immunoreactivities in the rabbit retina, respectively. Following ischaemia/reperjusion both the parvalbumin (A3) and ChAT (B3) immunoreactivities are reduced and clearly affected. However, treatment of animals with 8-0H-DPAT clearly blunted the effect of ischaemia/reperjusion, documented by a clear 'staining' for both parvalbumin (A2) and ChAT (B2).

L-type calcium channel46 it is concluded that the other type(s) of calcium channels, such as N-, P /Q- or T-. neuroprotective properties of 8-0H-DPAT and betaxolol Another possibility is that it acts directly at 5-HT lA are different. The possibility exists that 8-0H-DPAT receptors situated on the cortical neurones. However, reduces calcium influx into cortical neurones by acting at spiroxatrine (a putative 5-HT1A antagonist) did not blunt the effect of 8-0H-DPAT on the NMDA-induced influx 50 .------�------� of calcium, which does not support this idea (see Table 5). Thy.1 mRNA Rhodopsin mRNA '" .. '0 .. 25 �� 1.4 T---�======� c=J Control (n=9) �:i J.2 0.5% 8·0H·DPAT (n=4) 0 e,Sv ";jt' �S t .. ·25 =.� o� �! f;ol ·SO c=J NMDA � _ NMDA+ 8·0H·DPAT

.75 ...l...______-'- ______-.J

Fig. 8. The effect of intravitreal injection of N-methyl-D-aspartate (NMDA; at a finalconcentration of 200 pMin the vitreous; open bars) or NMDA plus 8-0H-DPAT (both 200 pM; shaded bars) on the overall levels of Thy-l and rhodopsin mRN A species in the retina o 2 5 (n = 4). Data are derived from densitometric analyses of duplicated RT-PCR experiments. Densitometric readings for Thy-l and rhodopsin Days mRNA species were calculated relative to cyclophilin mRNA levels and expressed as percentages of control amounts in each case. Note that Fig. 9. Summary of the effect of topically applied 0.5% 8-0H-DPAT rhodopsin mRNA is unaffected by NMDA whereas this compound on the amplitude of the b-wave of the electroretinogram (ERG) leads to a significant reduction in Thy-1 mRNA, which is partially following ischaemia. It can be seen that the b-wave recovery of ameliorated by 8·0H·DPAT. *p < 0.05, compared with control vehicle untreated animals following ischaemia and reperjusion times of 2 and 5

injected values using paired Student's t·test (n = 4); **p < 0.05, days is around 15%. In the 8-0H-DPAT treated animals the recovery compared with values obtained for NMDA-treated retinas using paired at 2 days is around 30% and at 5 days greater than 50%. *p < 0.05

Student's t-test (n = 4). compared with t = 0; **p < 0.05 compared with control.

460 c::::::::J (lOnM) c::::::::J 8-0H-DPAT _ betaxolol (100IlM)

100 � c .... e 80 '" c 'Q. :a c 60 '" .. ... Z s: 40 "2...., .... 0 OJ) c :a 20 c :Q �

I/lM 10llM 100/lM ImM

drug

Fig. 11. Effect of 8-0H-DPAT on the specific binding of 0.1 nM [3H]nitrendipine to rat cortical membranes. Effectsof nifedipine and betaxolol are shown for comparison. Each point represents the mean :!: SEM of three to five experiments performed in duplicate.

the pathophysiology of ischaemic damage. There are three major types of VSSCs in the brain, termed Rat I, Rat II and Rat III, which are responsible for the generation of action potentials in excitable membranes. Several neurotoxins are known to bind with high affinity to specific sites on these VSSCs and alter channel function. Five classes of these neurotoxin binding sites are Fig. 10. (A) Distribution of ChAT immunoreactivity in the rat retina. Immunoreactivity is associated with two bands of fibres in the inner currently recognised (neurotoxin sites 1-5), the most plexiform layer (small arrows) and perikarya on each side (large important of which are sites 1 and 2. Site 1 is located in arrows). Following ischaemia/reperjusion most of the ChAT the vestibule of the channel at the selectivity filter and immunoreactivity is obliterated (B). However, when animals are binding of toxins such as tetrodotoxin and saxitoxin treated topically with 8-0H-DPAT, the ischaemia/reperfusion-induced directly blocks the passage of Na + ions through the effect on the ChAT immunoreactivity is partially blunted with some ChAT 'staining' still apparent (C). channel. Conversely, site 2 is found in the transmembrane region and is involved in the gating of Recent studies have shown that substances which the channel. Lipid-soluble toxins including veratridine reduce sodium influx into neurones are also and batrachotoxin bind at site 2 causing a persistent neuroprotective.54 The neuroprotection may occur by activation of the sodium channel, which can be reversed indirectly reducing calcium influx; nevertheless, good by tetrodotoxin in a non-competitive fashion. Several evidence exists which demonstrates that activation of local anaesthetics, antiarrhythmics and anticonvulsants voltage-sensitive sodium channels (VSSCs) is involved in inhibit neuronal excitability by interacting with VSSCs at

Table 5. The effectof 8-0H-DPAT and 8-0H-DPAT plus spiroxatrine on the NMDA-induced accumulation of radioactive calcium by rat cortical cultures

Treatment 45Ca2+ uptake % in comparison with control values Control 100 NMDA (100 flM) 230 :!: 40 (n = 14) NMDA + 8-0H-DPAT (10 fl.M) 210 :!: 38 (n = 5) NMDA + 8-0H-DPAT (100 fl.M) 148 :!: 29 (n = 9) NMDA + 8-0H-DPAT (100 fl.M) + spiroxatrine (10 fl.M) 152 + 34 (n = 6) NMDA + MK-801 (5 fl.M) 114 + 11 (n = 4) Results are mean:!: SEM, each carried out in triplicate. Number (n) of experiments is shown in parentheses. Control cultures accumulated approximately 9000 dpm of 45Ca2+. Note: 8-0H-DPAT at 10 fl.M had no significant effect on the NMDA response.

461 100 tolerated by the patient as well as reaching the retina --- f1esinoxan when applied topically then the criteria for the ideal 90 -0- 8.0H·DPAT � glaucoma drug are approached. c 80 = Many 5-HTlA agonists are used in the treatment of '-'.... � 70 depression so they can be reasonably tolerated by patients. Moreover, 5-HT A agonists such as urapidil are � 60 1 =: known to dilate blood vessels, so theoretically could 50 ff'L.,. stimulate ocular blood flow. Our studies have shown that .... Q 40 IltI 5-HT1A agonists (8-0H-DPAT and flesinoxan) reduce C ] 30 lOP in rabbits. Moreover, we show that 8-0H-DPAT blunts the effects of insults (NMDA toxicity, ischaemia/ :E 20 � reperfusion) to the retinas of animals. The 10 neuroprotective effect of 8-0H-DPAT involves reducing 0 the influx of sodium but it may also involve interaction with retinal 5-HTIA and/or 5-HT7 receptors, although ·8 ·7 ·6 ·5 ·4 ·3 ·2 this remains to be demonstrated. Thus it is proposed that log [drug] (M) 5-HTlA agonists need to be considered as a class of drugs for the treatment of glaucoma. Fig. 12. Effect of 8-0H-OPAT and jlesinoxan on the specific binding of 10 nM [3H]batrachotoxinin (BTX) to rat forebrain membranes. Each point represents the mean ::': SEM of three experiments performed in duplicate. References 1. Quigley HA. Neuronal death in glaucoma. Prog Retin Eye Res 1999;18:39-57. neurotoxin site 2. More importantly, neuroprotective 2. Glovinsky Y, Quigley HA, Pease ME. Foveal ganglion cell compounds have also been shown to interact with loss is size dependent in experimental glaucoma. Invest neurotoxin site 2 in such a way as to inhibit Na + influx Ophthalmol Vis Sci 1993;34:395--400. induced by veratridine or batrachotoxin.55 The capacity 3. Morgan JE. Selective cell death in glaucoma: does it really for 5-HTIA agonists to interact with neurotoxin site 2 and occur? Br J Ophthalmol 1994;78:875-9. 4. Dreyer EB, Zurakowski D, Schumer RA, Podos SM, Lipton potentially to reduce sodium influx was therefore SA. Elevated glutamate levels in the vitreous body of investigated. humans and monkeys with glaucoma. Arch Ophthalmol In order to ascertain whether 5-HTIA agonists interact 1996;114:299-305. with VSSCs, radioligand binding experiments were 5. Osborne NN, Ugarte M, Chao M, Chidlow G, Bae JH, Wood JPM, et al. Neuroprotection in relation to retinal ischaemia performed using the neurotoxin site 1 and 2 selective 3 and relevance to glaucoma. Surv OphthalmoI1999;43:S102- ligands eH]saxitoxin and [ H]batrachotoxinin, 28. respectively. 8-0H-DPAT and flesinoxan had little effect 6. Louzada Junior P, Dias JJ, Santos WF, Lachat JI, Bradford HF, on the binding of [3H]saxitoxin to rat forebrain Coutinho Netto J. Glutamate release in experimental membranes (data not shown). However, both ischaemia of the retina: an approach using microdialysis. 3 J Neurochem 1992;59:358-63. compounds potently displaced [ H]batrachotoxinin 7. Neal MI, Cunningham JR, Hutson PH, Hogg J. Effects of binding, indicating that the drugs do interact with ischaemia on neurotransmitter release from the isolated neurotoxin site 2 (Fig. 12). Our latest preliminary data retina. J Neurochem 1994;62:1025-33. indicate that this interaction is inhibitory, since the influx 8. Vorwerk CK, Lipton SA, Zurakowski D, Hyman BT, Sabel 22 BA, Dreyer EB. Chronic low-dose glutamate is toxic to retinal of Na + into rat cortical synaptosomes induced by ganglion cells. Toxicity blocked by . Invest veratridine was dose-dependently attenuated by both 8- Ophthalmol Vis Sci 1996;37:1618-24. OH-DPAT and flesinoxan (data not shown). 9. Osborne NN, Chidlow G, Nash MS, Wood JPM. The potential of neuroprotection in glaucoma treatment. Curr Opin Ophthalmol 1999;10:82-92. 10. Osborne NN. 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