title: Pirenzepine affects scleral metabolic changes in myopia through a non-toxic mechanism.
abstract: Whilst the precise mechanism regulating ocular growth is unknown, it has been shown that various pharmacological agents, including the muscarinic receptor antagonists, atropine and pirenzepine, are effective at preventing the development of myopia. A recent study, which demonstrated that muscarinic antagonists reduce the synthesis of glycosaminoglycans and DNA in chick sclera in vitro, led to the suggestion that such drugs may act directly on the sclera, possibly through a toxic mechanism. Accepted markers of scleral metabolism and cell viability were used in conjunction with a non-invasive, physiological method of ocular growth regulation to determine whether the selective muscarinic antagonist pirenzepine inhibits the development of myopia via toxicity to the sclera. Chicks were monocularly deprived (MD) of pattern vision and given daily intravitreal injections of either pirenzepine (700 microg) or saline vehicle into the deprived eye over 5 days. Unoccluded animals also received intravitreal injections of either pirenzepine or saline into one eye (n=6, all groups). The contralateral eye of all animals was left untreated for comparison. Optical and ocular biometric measures were collected on the final experimental day. Following in vivo delivery of [(35)S] labelled sulphate, levels of sulphate incorporation into scleral glycosaminoglycans were measured in proteinase K digests following selective precipitation with alcian blue dye. The DNA content was also assessed through luminescence spectrometry after binding to Hoechst 33258 dye. To allow comparison with an accepted non-invasive, physiological method of ocular growth regulation, myopia was prevented in additional groups of MD animals by allowing 3hr of unoccluded vision each day, over 5 days, before levels of sulphate incorporation were measured. Scleral DNA content, a marker of cell viability, was not significantly altered between treated and control eyes in any injected group. Relative levels of sulphate incorporation (% difference between treated and contralateral control eyes) were significantly lower in the cartilaginous sclera of pirenzepine-MD animals, compared to saline-MD controls (+35.9 +/- 10.1% vs +121.2 +/- 28.6%, P<0.05), after 2hr of incorporation. However, after 6hr incorporation, there was no significant difference in sulphate incorporation in the cartilaginous sclera between the two groups (+87.2 +/- 33.1% vs +111.0 +/- 14.4%, P=0.53). No significant change was found in the levels of glycosaminoglycan synthesis in the fibrous sclera of any pirenzepine treated group, when compared to the appropriate saline control. Relative patterns of sulphate incorporation, between treatment and control groups, were essentially identical at both time points examined, regardless of whether myopia was prevented through pirenzepine injection or periods of unoccluded vision. The present study shows that, at a dose of pirenzepine sufficient to prevent experimentally-induced axial myopia, glycosaminoglycan synthesis in the cartilaginous sclera was significantly reduced for a transient period following the injection. These pirenzepine-induced reductions in glycosaminoglycan synthesis were not caused by direct drug toxicity to scleral cells as these changes were reversible and no significant reduction in DNA content was observed in pirenzepine treated eyes. Similar patterns of scleral glycosaminoglycan synthesis changes were found following the provision of brief periods of unoccluded vision further demonstrating that pirenzepine is effective in myopia prevention via a non-toxic mechanism. Consequently, the prevention of myopia development in chicks, with either pirenzepine or brief periods of unoccluded vision, is associated with the transient modulation of scleral glycosaminoglycan synthesis in the cartilaginous sclera.
authors.affiliation: Department of Optometry and Vision Sciences, The University of Melbourne, Victoria, 3010, Australia.
authors.forename: Hue-Trung
authors.initials: HT
authors.lastname: Truong
authors.forename: Charles L
authors.initials: CL
authors.lastname: Cottriall
authors.forename: Alex
authors.initials: A
authors.lastname: Gentle
authors.forename: Neville A
authors.initials: NA
authors.lastname: McBrien
mesh_terms: D000818:Animals; D001699:Biometry; D002470:Cell Survival / Q000187:drug effects; D002645:Chickens; D004247:DNA / Q000032:analysis; D006025:Glycosaminoglycans / Q000096:biosynthesis; D018727:Muscarinic Antagonists / Q000494:pharmacology*; D009216:Myopia / Q000378:metabolism* / Q000473:pathology / Q000517:prevention & control; D010890:Pirenzepine / Q000494:pharmacology*; D012029:Refraction, Ocular / Q000187:drug effects; D012590:Sclera / Q000187:drug effects* / Q000378:metabolism; D012683:Sensory Deprivation; D013431:Sulfates / Q000378:metabolism; D013997:Time Factors; D014822:Vitreous Body / Q000473:pathology
chemical_list: D006025:Glycosaminoglycans; D018727:Muscarinic Antagonists; D013431:Sulfates; D010890:Pirenzepine; D004247:DNA