This modular protocol makes it possible to explore multiple molecular events rendered by structural and signaling lipids, and/or RNAs, in discreet tissue regions and in blood with an improved quantitative and qualitative data output per sample specimen. The method is applicable to any experimental model, and it can also be applied to human tissue specimens and blood. Demonstrating the procedure will be Claudia Schwitter, a technician, Julia Post, a grad student, and Raissa Lerner, a postdoc in my group.
To begin, take previously-isolated, whole, frozen brains of mice with acute and prophylactically-treated kainic acid-induced epilepsy. Mount the brains onto the mounting system of the cryostat. Set the thickness to 50 micrometers and slice the brain in trim mode close to the region of interest.
When approaching the region of interest, set the thickness to 18 to 20 micrometers. To localize the subregion of interest, stain brain slices using toluidine blue. Use a microscope to inspect the stained slices to identify the regions of interest to be punched, and use a mouse atlas as reference to find the appropriate brain anatomical regions.
Use sample corers to take 0.8 to 1.0 millimeter-diameter punches. Transfer the punches for endocannabinoids and eicosanoids coextractions into pre-cooled 2 milliliter amber tubes with seven pre-cooled steel balls per tube. For phospholipids and endocannabinoids coextraction, dual lipid and RNA co-extraction, transfer the punches into 2 milliliter RNase-free extraction tubes with ceramic beads.
Weigh frozen punches in the cold room and immediately proceed with extraction or freeze at 80 degrees Celsius. To perform liquid-liquid lipid coextraction of endocannabinoids and eicosanoids from brain pieces, punches, or tissue powder samples, place the extraction tubes containing the tissue samples and seven steel balls on ice. Add 600 microliters of ice-cold MTBE and 50 microliters of acetonitrile water containing the internal standards.
After adding 400 microliters of 0.1 molar formic acid, use a tissue lyser to homogenize for 30 seconds to 1 minute. Centrifuge this homogenate for 15 minutes at 5, 000 times g at 4 degrees Celsius. Place it in the freezer for 10 minutes at 80 degrees Celsius to freeze the aqueous lower phase, which will ease the transfer of the upper organic phase.
Transfer the upper organic phase into new 1.5 milliliter amber tubes, evaporate under a gentle stream of nitrogen at 37 degrees Celsius for 10 minutes, and then reconstitute in 50 microliters of acetonitrile water for further analysis. Store the aqueous phase at 20 or 80 degrees Celsius for further protein content analysis. To coextract endocannabinoids and eicosanoids from plasma samples, place the frozen plasma samples on ice and let them completely thaw.
Add 800 microliters of MTBE and 50 microliters of acetonitrile water containing the internal standards optimized for spiking using reference plasma samples. Add 600 microliters of 0.1 molar formic acid and vortex at 4 degrees Celsius for 2 minutes. Centrifuge the samples at 4, 000 times g for 15 minutes at 4 degrees Celsius.
Transfer the organic phase into new tubes. Evaporate it under a gentle stream of nitrogen at 37 degrees Celsius, and then reconstitute it with 50 microliters of acetonitrile water for liquid chromatography multiple reaction monitoring analysis. To perform coextraction of phospholipids and endocannabinoids from brain regions, punches, or other tissue powder samples, place the extraction tubes containing the tissue samples and ceramic beads on ice.
Add 800 microliters of methyl tert-butyl ether and methanol mixture and 10 microliters of methanol containing the internal standards. Then, add 200 microliters of 0.1%formic acid containing 25 micromolar tetrahydrolipstatin/URB597 and 50 micrograms per milliliter of BHT. After homogenizing with a tissue homogenizer, centrifuge at 5, 000 times g and 4 degrees Celsius for 15 minutes.
Transfer the upper organic phase into new tubes, evaporate under a gentle stream of nitrogen at 37 degrees Celsius, and then reconstitute this lipid extract with 90 microliters of methanol. If proceeding immediately, add 10%water to an aliquot of lipid extract and inject 10 microliters in the LC/MS for phospholipid analysis. For further endocannabinoid analysis, proceed with the next step.
Take an aliquot of the lipid extract, evaporate to dryness, and reconstitute in acetonitrile water. To perform dual extraction of RNA and lipids and coextraction of phospholipids and endocannabinoids from tissue samples, thaw tissue powder aliquots or brain bunches at 4 degrees Celsius. Add the tissue to extraction tubes with ceramic beads.
Then add 600 microliters of RLT buffer together with 200 microliters of chloroform. For phospholipid and endocannabinoid coextraction, spike samples with 10 microliters of internal standard mixture and homogenize at high speed for 20 seconds. Transfer homogenized samples into new centrifuge tubes and centrifuge for five minutes at full speed and 4 degrees Celsius to enable the phase separation.
Transfer the upper phase to a fresh tube for RNA extraction using a standard kit. Elute extracted RNA in a total volume of 50 microliters of RNase-free water and store it at 80 degrees Celsius. For lipid extraction, add 800 microliters of MTBE/methanol and 200 microliters of 0.1%formic acid to the lower chloroform-containing phase.
Vortex for 45 minutes at 4 degrees Celsius. Transfer the upper organic phase to a new tube. Evaporate it under a gentle stream of nitrogen at 37 degrees Celsius.
For further LC/MS analysis, reconstitute the sample in 90 microliters of methanol and store it at 20 or 80 degrees Celsius, or proceed immediately to analysis. For further endocannabinoid analysis, proceed with the next step. Take an aliquot of the lipid extract, evaporate to dryness, and reconstitute in acetonitrile water.
Then, inject 20 microliters in the LC/MS for endocannabinoid analysis. Kainic acid induction of acute epileptic seizures led to a maximum seizure intensity one hour post-injection. Lipidomic profiling of endocannabinoids and eicosanoids, as well as phospholipids and endocannabinoids, shows lipid level changes across the cerebral cortex, striatum, thalamic region, hippocampus, hypothalamus, cerebellum as well as heart and lung tissue in kainic acid-induced epileptic mice and controls.
After dual extraction of phospholipids and endocannabinoids and RNA for quantitative profiling of mouse brain punches, the quantitative distribution of lipids in the hypothalamus, basolateral amygdala, and the ventral and dorsal hippocampus was analyzed. Relative expression levels of endogenous enzymes and receptors involved in lipid signaling, as well as markers for brain activity investigated at the mRNA level in different brain regions and subregions from mice subjected to kainic acid-induced epileptic seizure and controls. Kainic acid-induced epilepsy caused massive loss of NeuN signal, predominantly in the CA1, CA3, and hilus region of the hippocampus, accompanied by apoptotic events indicated by caspase-3 signal compared to the control saline-injected mice.
After treatment with subchronic palmitoylethanolamide, neuronal nuclei protein signal was noticeably preserved and CASP3 signal was barely detectable. Brain punching and dissection of discreet brain areas are quite challenging and require fine skills to obtain consistent sampling across multiple cohorts. Therefore, practicing on test organs and good knowledge of brain morphology are highly recommended.
