The time course
of changes in mRNA levels in chick sclera during induced myopia
and hyperopia development
In birds, the increased ocular elongation caused
by form deprivation and minus lens wear is associated with increased
incorporation of precursors into DNA, protein, and glycosaminoglycans
in the sclera.
The chick sclera is composed of two layers: an outer fibrous layer
(like that in mammals) and an inner cartilaginous layer. The tissue
remodelling in the sclera during increased eye growth can involve
a number of extracellular matrix (ECM) proteins and enzymes. These
include structural components, such as collagen, degradative enzymes,
such as the matrix metalloproteinases (MMPs) that degrade ECM proteins
and tissue inhibitors of metalloprotinases (TIMPs) that bind to
and inhibit the activity of the MMPs.
The levels of many proteins, including MMPs, are significantly influenced
at the level of transcription.
Fig. 1: Section of the posterior wall of a
normal chick eye (14 days old), showing the two layers of sclera.
FS = fibrous sclera; CS = cartilaginous sclera, CH = choroid.
During recent studies, we found out that the
mRNA levels of some retinal genes is coupled in both eyes, even
if only one eye is treated with a lens. Only the lens treated eye
changes its growth.
Ruth Schippert would like to find out where the signalling cascades in the treated and untreated eyes differ. Therefore, chicks were monocularly treated with lenses and occluders for different time points.
She separates the two different scleral layers and compares the
mRNA levels of aggrecan, collagen, TIMP-2, MMP-2 and TGF-ß2 in the
treated eyes and the contralateral control eyes.
Fig. 2: Relative gene expression of aggrecan,
MMP-2, TIMP-2, TGF-ß2 and 18S-rRNA in chicken cartilaginous sclera.
Refractive development and eye growth in a mouse mutant lacking the transcription factor Egr1
Since an increase in ZENK mRNA and ZENK immunoreactivity in the retina was observed when hyperopia is induced in chickens, and a decline in ZENK is observed when myopia develops, Ruth Schippert studied a knock-out mouse which lack functional ZENK (in mammals called Egr1). The hypothesis was that this mouse would have longer eyes and be more myopic.
Using the optical low coherence interferometry (the Zeiss ACMaster - see Christine Schmucker), photorefraction (see Frank Schaeffel), photokeratometry (see Christine Schmucker) and optomotor testing
(see Christine Schmucker), Ruth showed that these mice indeed have longer eyes, at least between day 25 and 45 post-hatching. They were also more myopic, but showed no deficits in optomotor testing. This study was published in IOVS.
Effects of peripheral and central defocus on refractive development in the chicken
Effects of peripheral and central defocus on refractive development in the chicken
The laboratory of Professor Earl Smith has shown that refractive errors can be induced in monkeys if only the peripheral retinal image is degraded or defocused. The fovea may have had normal vision. Since this finding may have implications on the future design of spectacle lenses, this finding is of particular importance.
Ruth has used spectacle lenses with holes in their centres of various diameters. Through these holes, the chicks could see normally in the centre of the visual field, but the periphery was defocused. Interestingly, the chicks could compensate the defocus very locally, as can be seen in Figures 3-5.
This study was published in Vision Research in 2006: Schippert R, Schaeffel F. Peripheral defocus does not necessarily affect central refractive development Vision Res. 2006 Oct;46(22):3935-40
Fig. 3: Local compensation of refractive state after treatment with lenses which had holes of different diameters (0-8 mm) in their centres.
Fig. 4: The growth pattern induced in the chicken eye during the treatment with lenses with holes in their centres.
Since no "perfect" target was found yet for a pharmacological intervention of myopia development, several laboratories have initiated screenings with microarrays - little plates on which the short DNA sequences are densely spotted (up to 30000 on one plate) and to which DNA extracts from the tissue under investigation can bind.
The strength of binding can be quantified because the fragments are labelled with fluorescent dyes. After treating animals with lenses or diffusers, mRNA is extracted from the retina, RPE, choroid or sclera reverse transcribed and bound to the microarray. A control is usually the untreated fellow eye.
Unfortunately, these experiments provide many new candidates, and the most confusing observation is currently that different labs obtain different candidates. This traces back to minor differences in the strains of animals or treatment conditions, and it becomes clear that the treatments needs to be matched as closely as possible. Another problem is that usually the complete retina is extracted, and that changes in different cell types may be in opposite directions and cancel each other out. Therefore, a cell- or at least a layer-specific analysis becomes necessary
(see Regan Ashby).
Ruth has performed analyses with chick microarrays (currently under revision at Molecular Vision) and in the EGR1 knock-out mouse (see project 2, above) - these data are presented at ARVO 2008.
