Detection of small molecule concentration gradients in ocular tissues and humours

Abstract

The eye is an elegant organ consisting of a number of tissues and fluids with specialised functions that together allow it to effectively transmit and transduce light input to the brain for visual perception. One key determinant of this integrated function is the spatial relationship of ocular tissues. Biomolecular distributions within the main ocular tissues cornea, lens, and retina have been studied extensively in isolation, yet the potential for metabolic communication between ocular tissues via the ocular humours has been difficult to visualise. To address this limitation, the current study presents a method to map spatial distributions of metabolites and small molecules in whole eyes, including ocular humours. Using a tape-transfer system and freeze-drying, the spatial distribution of ocular small molecules was investigated in mouse, rat, fish (black bream), and rabbit eyes using negative ion mode MALDI imaging mass spectrometry. Full-scan imaging was used for discovery experiments, while MS/MS imaging for identification and localisation was also demonstrated. In all eyes, metabolites such as glutathione and phospholipids were localised in the main ocular tissues. In addition, in rodent eyes, major metabolites were distributed relatively uniformly in ocular humours.In contrast, both uniform and spatially defined ocular metabolite distributions were observed in the black bream eye. Tissue and ocular humour distributions were reproducible, as demonstrated by the three-dimensional analysis of a mouse eye, and able to be captured with high spatial resolution analysis. The presented method could be used to further investigate the role of inter-tissue metabolism in ocular health, and to support the development of therapeutics to treat major ocular diseases.

Partitioning and Spatial Distribution of Drugs in Ocular Surface Tissues

“…Previously, only a few MALDI-IMS studies on in vivo ocular pharmacokinetics of atropine [ 52 , 53 ] and brimonidine [ 54 ] have been published. In addition, MALDI-IMS was used earlier to analyze the distribution of several drugs in isolated lenses [ 55 ] and to detect endogenic metabolites and lipids in the eye [ 47 ]. MALDI-IMS is a powerful analytical technique that allows studying the spatial distribution of several drugs in more than one tissue at a time and providing higher spatial resolution than traditional LCMS/MS methods.…”

“…The samples were sectioned to 20 μm or 35 μm thickness at −20 °C on a Leica CM Cryostat (S3050, Leica Microsystems GmbH, Wetzlar, Germany) and collected on a cryo-film (3C16UF, SECTION-LAB Co. Ltd., Yokohama, Japan) by a modified Kawamoto method [ 46 ]. The film was subsequently mounted onto a microscope glass slide using a double-sided copper tape, and the sample slides were dried in a lyophilization chamber [ 47 ]. The slides were washed twice with 50 mM ammonium formate for 10 s and dried in a vacuum desiccator.…”

Age‐dependent changes in glutathione metabolism pathways in the lens: New insights into therapeutic strategies to prevent cataract formation—A review

“…Perhaps more interestingly, it has also allowed the direct comparison of the spatial distributions and relative abundances of other GSH-related molecules within the same tissue sections, demonstrating the potential for spatial mapping of GSH metabolism pathways. This may be an interesting approach 94 to utilize in future studies designed to understand the role of the lens in maintaining ocular health as we age.…”

Mapping glucose metabolites in the normal bovine lens: Evaluation and optimisation of a matrix‐assisted laser desorption/ionisation imaging mass spectrometry method

“…While IMS was first developed to map protein and peptide distributions, more recently, it has been used to map small molecules and metabolites and is particularly powerful in this area when coupled to mass analysis via Fourier transform‐ion cyclotron resonance (FT‐ICR) 20 . A range of lens metabolite classes have been mapped using MALDI‐FT‐ICR IMS 21–23 . Several of the glucose metabolites have been detected in other tissues using MALDI matrices, 24,25 and isobaric hexose monophosphates and bisphosphates imaged elegantly in the brain by using MS 3 on a linear ion trap 26 .…”

“…20 A range of lens metabolite classes have been mapped using MALDI-FT-ICR IMS. [21][22][23] Several of the glucose metabolites have been detected in other tissues using MALDI matrices, 24,25 and isobaric hexose monophosphates and bisphosphates imaged elegantly in the brain by using MS 3 on a linear ion trap. 26 However, the tissue preparation and MALDI matrix conditions for glucose and glucose metabolite detection in the lens have not previously been investigated.…”