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

While decades of
technical and analytical advancements have been
utilized to discover novel lipid species, increase speciation, and
evaluate localized lipid dysregulation at subtissue, cellular, and
subcellular levels, many challenges still exist. One limitation is
that the acquisition of both in-depth spatial information and comprehensive
lipid speciation is extremely difficult, especially when biological
material is limited or lipids are at low abundance. In neuroscience,
for example, it is often desired to focus on only one brain region
or subregion, which can be well under a square millimeter for rodents.
Herein, we evaluate a micropunch histology method where cortical brain
tissue at 2.0, 1.25, 1.0, 0.75, 0.5, and 0.25 mm diameter sizes and
1 mm depth was collected and analyzed with multidimensional liquid
chromatography, ion mobility spectrometry, collision induced dissociation,
and mass spectrometry (LC-IMS-CID-MS) measurements. Lipid extraction
was optimized for the small sample sizes, and assessment of lipidome
coverage for the 2.0 to 0.25 mm diameter sizes showed a decline from
304 to 198 lipid identifications as validated by all 4 analysis dimensions
(∼35% loss in coverage for ∼88% less tissue). While
losses were observed, the ∼200 lipids and estimated 4630 neurons
contained within the 0.25 punch still provided in-depth characterization
of the small tissue region. Furthermore, while localization routinely
achieved by mass spectrometry imaging (MSI) and single cell analyses
is greater, this diameter is sufficiently small to isolate subcortical,
hypothalamic, and other brain regions in adult rats, thereby increasing
the coverage of lipids within relevant spatial windows without sacrificing
speciation. Therefore, micropunch histology enables in-depth, region-specific
lipid evaluations, an approach that will prove beneficial to a variety
of lipidomic applications.