X-ray fluorescence microscopic measurement of elemental distribution in the mouse retina with age

Abstract

The biologically important metals such as zinc, copper and iron play key roles in retinal function, yet no study has mapped the spatio-temporal distribution of retinal biometals in healthy or diseased retina. We investigated a natural mouse model of retinal degeneration, the Cln6 mouse. As dysfunctional metabolism of biometals is observed in the brains of these animals and deregulated metal homeostasis has been linked to retinal degeneration, we focused on mapping the elemental distribution in the healthy and Cln6 mouse retina with age. Retinal and RPE elemental homeostasis was mapped in Cln6 and C57BL6/J mice from 1 to 8 months of age using X-ray Fluorescence Microscopy at the Australian Synchrotron. In the healthy retina, we detected a progressive loss of phosphorus in the outer nuclear layer and significant reduction in iron in the inner segments of the photoreceptors. Further investigation revealed a unique elemental signature for each retinal layer, with high areal concentrations of iron and sulfur in the photoreceptor segments and calcium, phosphorus, zinc and potassium enrichment predominantly in the nuclear layers. The analysis of retinae from Cln6 mice did not show significant temporal changes in elemental distributions compared to age matched controls, despite significant photoreceptor cell loss. Our data therefore demonstrates that retinal layers have unique elemental composition. Elemental distribution is, with few exceptions, stably maintained over time in healthy and Cln6 mouse retina, suggesting conservation of elemental distribution is critical for basic retinal function with age and is not modulated by processes underlying retinal degeneration.

Pathway analysis identifies altered mitochondrial metabolism, neurotransmission, structural pathways and complement cascade in retina/RPE/ choroid in chick model of form-deprivation myopia

“…The suppression in bile acid metabolism during early recovery from profound FDM highlights the importance of maintaining energy metabolism in myopia. The GSEA findings provide supporting evidence for the RIDE model as well as complementing earlier biometric and ultrastructural findings showing that form-deprivation occlusion leads to changes in eye volume, refraction, thinning of the retina and choroid, and morphological evidence for hyperosmolarity ( Brocker, Thompson & Vasiliou, 2012 ; Crewther et al, 2006 ; Grubman et al, 2016 ; Hollborn et al, 2017 ; Junghans et al, 1999 ; Liang et al, 1995 ; Liang et al, 2004 ). Although a combination of posterior ocular tissue have been analysed in previous transcriptome studies on refractive errors ( McGlinn et al, 2007 ; Rada & Wiechmann, 2009 ; Riddell et al, 2016 ; Shelton et al, 2008 ; Stone et al, 2011 ), commonalities in differentially expressed genes have now been identified regardless of species, tissue analysed and genomic platform ( Riddell & Crewther, 2017a ).…”

“…Previous ultrastructural studies of form-deprivation in chick have demonstrated morphological abnormalities in photoreceptor outer segments, retinal pigment epithelium (RPE) nuclei, mitochondria and basal laminae ( Beresford, Crewther & Crewther, 1998 ; Liang et al, 1995 ; Liang et al, 2004 ; Liang et al, 1996 ) similar to those described elsewhere as characteristic of AMD ( Datta et al, 2017 ), CNV ( Ohno-Matsui et al, 2017 ) and glaucoma ( Kim & Park, 2017 ). These morphological changes occur concomitantly with elemental microanalytical evidence of hyperosmotic changes in ion distribution patterns across the retina, RPE and choroid ( Crewther et al, 2006 ; Grubman et al, 2016 ; Hollborn et al, 2017 ; Junghans et al, 1999 ; Liang et al, 1995 ; Liang et al, 2004 ) and reminiscent of physiologically induced hyperosmotic and oxidative stress elsewhere in the brain ( Brocker, Thompson & Vasiliou, 2012 ; Morland, Pettersen & Hassel, 2016 ; Veltmann et al, 2016 ). Indeed, oxidative stress has been suggested to contribute to the underlying mechanisms involved in profound myopia pathology ( Francisco, Salvador & Amparo, 2015 ).…”

Peer Review #1 of "Pathway analysis identifies altered mitochondrial metabolism, neurotransmission, structural pathways and complement cascade in retina/RPE/ choroid in chick model of form-deprivation myopia (v0.1)"

“…The suppression in bile acid metabolism during early recovery from profound FDM highlights the importance of maintaining energy metabolism in myopia. The GSEA findings provide supporting evidence for the RIDE model as well as complementing earlier biometric and ultrastructural findings showing that form-deprivation occlusion leads to changes in eye volume, refraction, thinning of the retina and choroid, and morphological evidence for hyperosmolarity (Brocker et al 2012;Crewther et al 2006;Grubman et al 2016;Hollborn et al 2017;Junghans et al 1999;Liang et al 1995;Liang et al 2004). Manuscript to be reviewed Stone et al 2011), commonalities in differentially expressed genes have now been identified regardless of species, tissue analysed and genomic platform (Riddell & Crewther 2017a).…”

“…2017), CNV (Ohno-Matsui et al 2017) and glaucoma (Kim & Park 2017). These morphological changes occur concomitantly with elemental microanalytical evidence of hyperosmotic changes in ion distribution patterns across the retina, RPE and choroid (Brocker et al 2012;Crewther et al 2006;Grubman et al 2016;Hollborn et al 2017;Junghans et al 1999;Liang et al 1995;Liang et al 2004) and reminiscent of physiologically induced hyperosmotic and oxidative stress elsewhere in the brain (Brocker et al 2012;Morland et al 2016;Veltmann et al 2016). Indeed, oxidative stress has been suggested to contribute to the underlying mechanisms involved in profound myopia pathology (Francisco et al 2015).…”

Elemental characterisation of the pyramidal neuron layer within the rat and mouse hippocampus

“…The nominal sample composition and thickness estimate is used to provide a first-order self-absorption correction, and inaccuracies have negligible impact on the quantitative concentrations of the first-row transition metals in these thin tissue sections, as reported by several groups. [83][84][85] Lighter element such as S and P are presented for distributional purposes only; absolute quantification of these elements is difficult and subject to significant errors from the assumption of matrix composition, thickness and density. Quantitative data were extracted as tiff files of quantitative per-pixel elemental area density in ng cm À2 , which were then imported into ImageJ v1.48, as described previously.…”