Combining Micropunch Histology and Multidimensional Lipidomic Measurements for In-Depth Tissue Mapping

While decades oftechnical and analytical advancements have beenutilized to discover novel lipid species, increase speciation, andevaluate localized lipid dysregulation at subtissue, cellular, andsubcellular levels, many challenges still exist. One limitation isthat the acquisition of both in-depth spatial information and comprehensivelipid speciation is extremely difficult, especially when biologicalmaterial is limited or lipids are at low abundance. In neuroscience,for example, it is often desired to focus on only one brain regionor subregion, which can be well under a square millimeter for rodents.Herein, we evaluate a micropunch histology method where cortical braintissue at 2.0, 1.25, 1.0, 0.75, 0.5, and 0.25 mm diameter sizes and1 mm depth was collected and analyzed with multidimensional liquidchromatography, ion mobility spectrometry, collision induced dissociation,and mass spectrometry (LC-IMS-CID-MS) measurements. Lipid extractionwas optimized for the small sample sizes, and assessment of lipidomecoverage for the 2.0 to 0.25 mm diameter sizes showed a decline from304 to 198 lipid identifications as validated by all 4 analysis dimensions(& SIM;35% loss in coverage for & SIM;88% less tissue). Whilelosses were observed, the & SIM;200 lipids and estimated 4630 neuronscontained within the 0.25 punch still provided in-depth characterizationof the small tissue region. Furthermore, while localization routinelyachieved by mass spectrometry imaging (MSI) and single cell analysesis greater, this diameter is sufficiently small to isolate subcortical,hypothalamic, and other brain regions in adult rats, thereby increasingthe coverage of lipids within relevant spatial windows without sacrificingspeciation. Therefore, micropunch histology enables in-depth, region-specificlipid evaluations, an approach that will prove beneficial to a varietyof lipidomic applications.

Automated summary

This study assessed a micropunch histology method for analyzing brain tissue in rodents, demonstrating a decline in lipidome coverage from samples of 2.0 to 0.25 mm in diameter, but in-depth characterization was still achieved in the 0.25 mm sample. This approach enables region-specific lipid evaluations in small tissue regions, increasing coverage of lipids within relevant spatial windows without sacrificing speciation.