Muscarinic antagonists, atropine have been shown to reduce myopia development in the animal models of macaques Raviola and Wiesel, 1985, rhesus monkeys (Tigges et al., 1999), and chicks (Stone et al., 1991; McBrien et al., 1993). Furthermore, it was shown that atropine reduces axial myopia in chickens, in which cholinergic transmission to the ciliary muscle is nicotinic rather than muscarinic. A non-accommodative mechanism was proposed (McBrien et al., 1993). Pirenzepine (M1 and M4 selective) also shown to reduce the experimental myopia in chicks and tree shrew (Leech et al., 1995; Cottriall and McBrien, 1996; Cottriall et al., 1999). Recently, atropine and pirenzepine have been shown in clinical trails to slow the progression of myopia in children (Tan et al., 2005; Chua et al., 2006; Siatkowski et al., 2008). Furthermore, the M4 selective muscarinic antagonist, himabacine, was effective at reducing the excessive vitreous chamber elongation associated with myopia development and also demonstrated that no evidence of toxic damage to the retina (Cottriall et al., 2001b). Further studies utilising highly selective muscarinic antagonists from snake toxins have also implicated M4 and M1 receptors in mediating control of myopic eye growth in chick (Diether et al., 2005; Morgan et al., 2005). In an other experiment from our lab revealed, as chick does not posses an M1 muscarinic receptor, the Muscarinic Toxin 7 (MT7), a M1 selective antagonist are ineffective in the control of myopia in chicks, while Muscarinic Toxin 3 (MT3), a M4 selective antagonist inhibited myopia effectively in an dose-dependent manner. Furthermore, last experimental chapter has claimed that the both MT7 and MT3 are effective in the control of myopic ocular growth in tree shrews. These results confirm that the both M1 and M4 muscarinic receptors are involved in controlling myopia.
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On the other hand, dopamine, a neurotransmitter have been implicated in the control of myopia and it is consistently reported that the retinal level of dopamine and its principal metabolite 3,4-dihydroxyphenlacetic acid (DOPAC) reduced in form deprived myopia in chick (Stone et al., 1989), monkey (Iuvone et al., 1989) and in tree shrew (McBrien et al., 2001). It is synthesized and released by an amacrine or modified amacrine (interplexiform) cell, also called "DA amacrine cell"(Witkovsky and Schutte, 1991; Luft et al., 2004). In the retina, dopamine acts both as a conventional synaptic neurotransmitter and through diffusion, on more distant targets (Witkovsky et al., 1993). It was argued that the reduction of retinal dopamine synthesis and release during short period (2 hr) of form-deprivation is due to the reduction in light intensity, rather than the effects of a diffuser itself (Luft et al., 2004). However, reports have shown that effect of form deprivation on dopamine release was greater than that of a neutral density filter of equal transmittance after 5 days of diffuser treatment (Feldkaemper et al., 1999). Furthermore, the retinal dopamine and DOPAC is reduced following minus lens induced myopia, while hyperopia induced by the positive lenses both contents were increased after two weeks of treatment (Guo et al., 1995). Recently Megaw and colleagues (2001) suggested the use of vitreal DOPAC content as indices to infer the retinal dopamine release (Megaw et al., 2001), the idea has been explored from the past report (Witkovsky et al., 1993).
Apomorphine, a dopamine agonist (relative non selective) effective in the control of chick myopia in a dose dependent manner (Stone et al., 1989) the similar effect was shown soon after in primates (Iuvone et al., 1991). Studies have claimed that the dopaminergic myopia control mediated by D2- receptors (Rohrer et al., 1993; McCarthy et al., 2007). Furthermore, spiperone abolished the form deprived myopia blocking activity of apomorphine when both were injected intravitreally (Rohrer et al., 1993). McCarthy and colleagues (2007) have shown, quinpirole, a D2 specific agonist is effective in controlling form deprived myopia in chicks while spiperone, a D2 antagonist, having no effect on normal ocular growth and form deprived myopia (McCarthy et al., 2007). Previously it was found that deprivation myopia could also be suppressed by a neurotoxin of catecholaminergic cells (6-hydroxy dopamine), which inhibits the dopaminergic pathways in the retina (Li et al., 1992). Possible explanation of why 6-hydroxy dopamine (6-OHDA) suppresses deprivation myopia could due to partial depletion of the dopaminergic system in the retina, a state of hypersensitivity to dopamine such that a low dopamine level is sufficient to block the deprivation myopia (Schaeffel et al., 1994). 6-OHDA does not affect the lens induced myopia and proposed the idea of form deprivation and lens induced axial elongation in chicks may be based on pharmacologically different mechanisms (Schaeffel et al., 1994)
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Few studies have clearly demonstrated the existence of interactions between muscarinic and dopaminergic control of myopia, an indirect cholinomimetic, diisopropylfluorophosphate (DFP) expected to increase the level of myopia, instead it is effective in controlling form-deprived myopia compared to control eyes (-20.9 D vs -8.2 D). Surprisingly, retinal dopamine and DOPAC were reduced following 8 days of form deprivation (four DFP injections every other day), which is similar to control animals. Retinal dopamine content was also measured after the acute treatment with DFP, following 1.5 hr significant increase in the dopamine and DOPAC level. Finally it was argued that DFP induced changes in the dopamine system and suggested link between the cholinergic and dopaminergic systems. The use of spiperone along with DFP could account for 30% return to control group level of myopia and further concluded that change in the dopamine is not the only sole mechanism by which DFP controls the myopia (Cottriall et al., 2001a). Atropine causes a long lasting increase in dopamine release in vitro and a significant increase in vitreal dopamine 1 hr after intravitreal injection (Schwahn et al., 2000). In line with the successful of M1 and M4 muscarinic receptor control of myopia (previous chapter result), a D2 dopamine receptor has also been implicated in the control of myopia. Till to date there is no studies tested the close interactions between dopamine and muscarinic system using highly selective drugs to the respective receptor subtypes.
The present study sought to determine whether the M4 muscarinic receptor control of myopic ocular growth signal guided via the dopaminergic (D2 receptor) pathway.
Spiperone (D2 antagonist) in combination of MT3 will present the inhibition of myopia induced by muscarinic antagonist.
As chick does not have an M1 muscarinic receptor, would be ideal to use in this experiment. Four groups of chicks (n=8 in each group) will under go 10Âµl daily intravitreal (IV) injections along with monocular deprivation to their left eyes using velcro goggle. Right eyes will serve as control. Doses and groups are detailed in the table below.
Â Drugs / Doses
MT3 + spiperone
0.1 % Ascorbate
2.5ÂµM + 5ÂµM
Spiperone concentration were calculated based on their in vitro affinity (IC50) = 55nM (Martin et al., 1984) and Following 5 days treatment procedure, the refractive and structural measurement will be taken. Tissue also will be collected for histology to rule out any toxic damage to the retina.
Dopamine - Pros
Rule out the dopamine system involvement in the control of myopia and confirming the muscarinic pathway (M1 and M4)
Till to date no studies tested the close interactions between two systems using highly selective drugs.
Dopamine - Cons
Introducing the dopamine system to test the muscarinic control of myopia
Indirect cholinomimetic have also effective in controlling myopia and the use of spiperone could account for 30% return to control group level
Light level change itself changes the retinal dopamine and DOPAC content atleast for short duration (no difference between occluder and neutral density filter after 2 hr of treatment)
6-OHDA also inhibiting the myopia in form deprived chicks