Name: lipidomics and transcriptomics in neurological diseases
Uploaded: 2022-03-18T12:30:00
Description: Watch this Scientific Journal Video about Lipidomics and Transcriptomics in Neurological Diseases at JoVE.com

We dedicate this article to Dr. Ermelinda Lomazzo. During the finalization of this manuscript, Dr. Ermelinda Lomazzo passed away. She is the embodiment of passion for science and selfless engagement in team work to fulfill a meaningful research purpose. She always dreamed of contributing meaningfully to the greater well-being of humans. Her goodhearted nature was never compromised by the strenuous roads of science and life. She will remain invaluable, and forever, in our hearts.
Julia M. Post was funded by Focus Program for Translational Neuroscience (FTN) at University Medical Center of the Johannes Gutenberg University Mainz and is currently funded by the SPP-2225 EXIT project to LB. Raissa Lerner was partially funded by DZHK project 81X2600250 to LB and Lipidomics Core Facility. Partial funding for these studies was provided by the Lipidomics Core Facility, Institute of Physiological Chemistry, and Intramural funds (to LB) from the University Medical Center of the Johannes Gutenberg University Mainz.

All experimental procedures described here are in accordance with The European Community&#39;s Council Directive of 22 September 2010 (2010/63EU) and were approved by the local animal committee of the state Rhineland-Palatinate, Germany (file reference: 23 177-07/G16-1-075).
1. Animal model of acute and prophylactically treated KA-induced epilepsy
<ol>
	<li>Perform seizure induction, treatment, and behavioral scoring.
	<ol>
		<li>Separate mice (minimum n = 6 mice per group) in single cages.<...

<ol>
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E., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+determinants+of+susceptibility+to+excitotoxic+cell+death:+Implications+for+gene+targeting+approaches.">Genetic determinants of susceptibility to excitotoxic cell death: Implications for gene targeting approaches.</a> Proceedings of the National Academy of Sciences. 94 (8), 4103-4108 (2002).</li><li>Monory, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Endocannabinoid+System+Controls+Key+Epileptogenic+Circuits+in+the+Hippocampus.">The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus.</a> Neuron. 51 (4), 455-466 (2006).</li><li>Konsman, J. 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The neurolipidomic and transcriptomic methodology described here is a viable mean to investigate any disease or healthy development at high and low spatial resolution in the brain and peripheral organs. Due to the optimized plasma sampling and handling procedures, plasma lipidomic analysis can also be carried out from the same animals sacrificed for tissue lipidomics and transcriptomics, thus improving the reliability of tissue blood molecular correlates and biomarker discovery. The provision of a broad set of data by ap...

Recent advances in the function of lipids and their role in the onset and progression of neurological diseases open new research and development venues of new therapeutic targets and disease mechanism elucidation1. Documented differences in lipid composition in different brain regions, emphasized by modern molecular imaging techniques such as mass spectrometry imaging and advanced mass spectrometry profiling, shifts the paradigm of lipid investigation from whole brain toward functionally distinct and discrete brain regions. The fact that lipid composition varies in different brain regions prompts new conceptualization of both membrane lipid sensitivity and downstream lipid signaling in response to a brain insult or stimuli across the functionally distinct brain regions. Hence, lipid protocols require new developments to address the challenge of low tissue amounts for higher spatial resolution detection and quantification, and concurrently, analysis of multiple lipid components of cell membranes and signaling pathways. Also, determination of enzymes, lipid ligands, and receptors involved in the regulation of their levels and function is paramount to elucidate the signaling pathways affected in a neurological disease and guide new mechanistic investigations in a pathophysiological context.
In addition to the increased brain spatial resolution, there are two major difficulties challenging the development of new neurolipidomic approaches. First, the lipid signaling molecules are typically of very low abundance compared to membrane constitutive lipids. Second, the lipidome exhibits a high structural heterogeneity, difficult to dissect using a single analytical approach. Hence, extraction and analytical methods are tailored to different lipid categories and commonly performed in distinct tissue samples2. Shotgun lipidomic methods3 are excellent tools to rapidly reveal a broad profile of membrane lipids, while increased sensitivity and selectivity afforded by the targeted discovery and quantification mass spectrometric methods are capitalized upon for investigation of low abundant signaling lipids including: i) inflammatory lipids and ii) lipids involved in the modulation of neuronal activity, such as endocannabinoids (eCBs), amino acid-linked lipids, etc.4,5. To encompass lipid changes at both the cell membrane and&#160;signaling level occurring in brain regions of neurological disease models, typically the lipid extraction and analysis are carried out in distinct tissue samples, obtained from distinct animal batches or from different hemispheres, or by dissecting a larger tissue region into multiple pieces. When mRNA levels of enzyme receptors are also of interest, their investigation typically requires the procurement of a distinct tissue sample. For example, the investigation of membrane lipids, endogenous cannabinoids, and mRNA would require three different tissue samples, (e.g., two samples for the two lipid extraction methods-membrane lipids and signaling lipids- and subsequent two lipid analysis methods- and one sample for mRNA analysis). Investigation of inflammatory lipids and endogenous cannabinoids require two distinct tissue samples, extraction methods, and analysis methods, respectively. Another example is the investigation of mRNA and of any lipid category in a brain punch or laser microdissection sample which consequently requires two distinct animals to procure two samples per brain (sub)region. A substantial extent of variability and/or poor reproducibility of the results frequently occur in such cases, originating from biological variability and/or tissue heterogeneity. Guided by these practical limitations of multimolecular analysis, occurring particularly at high spatial resolution in the brain, a three-module neurolipidomics protocol was designed encompassing: 1) coextraction and co-analysis by LC/MRM of inflammatory lipids (e.g., eicosanoids (eiCs)) and lipids involved in modulation of neuronal activity, such as eCBs2; 2) co-extraction of phospholipids (PLs) and eCBs with subsequent multiscan LC/MRM and precursor/neutral loss scan analysis2; and 3) dual extraction of membrane (phospho)lipids and eCBs as well as mRNA, with subsequent LC/MRM and qPCR or RNA sequencing analysis6. Depending on the biological question to be addressed in a neurological disease and the brain region of interest, a combination of the first and the second protocol, or the first and the third protocol, can be applied on the same tissue specimen for tissues weighing around 4 mg. The first and third protocols can be independently applied for tissues around 2 mg. The second protocol can be applied for tissues weighing as little as 0.5 mg. Irrespective of the neurolipidomic protocol module selected, the tissue sampling and pre-analytical processing, the brain isolation and region dissection, as well as the procedure for sacrificing the animal model are standardized and identical for all three modules of the protocol. In our investigation of neurological diseases, peripheral organs that are relevant for the pathological consequences of the disease are always also collected and analyzed using these modular protocols. Additionally, blood is regularly sampled for plasma lipidomics to serve as a readout tool of neurological diseases with a view on prospective translational applications. The here presented modular lipidomics protocol is very versatile: scaleable to larger tissue amounts and readily applicable for virtually any tissue type and disease. For the application of the modular protocol (Figure 1) in neurological diseases, any standardized rodent model of onset and progression of neurological disorders, such as traumatic brain injury, Parkinson&#39;s disease, Alzheimer&#39;s disease, or epilepsy are amenable.
These protocols have been extensively applied to study changes in the tissue lipidome and/or transcriptome at the acute phase of epilepsy in the kainic acid (KA)-induced mouse model of epilepsy2,7, a model widely used in preclinical studies due to the resemblance to human temporal lobe epilepsy (TLE)8,9,10,11. Using these protocols, the therapeutic potential of drugs such as Palmitoylethanolamide (PEA)12,13 was assessed in the same mouse model of epilepsy. The study identified lipid and mRNA changes at high and low spatial resolution in the brain and periphery, at the time point of maximal acute seizure intensities (at 60 min postseizure induction), and upon subchronic and acute treatment with PEA at four different timepoints (20, 60, 120, and 180 min) post KA-seizure induction, a time-window covering the acute phase of epilepsy. Plasma, brains, and peripheral organs of untreated KA-injected mice, acute and subchronically PEA-treated mice, as well as vehicle and PEA-vehicle control mice, were collected at each time point12,13, and investigated with this molecular analysis. The molecular data were correlated with behavioral phenotypes obtained by seizure scoring, as well as with immunohistochemistry-derived data on neurodegenerative processes, in order to unravel the progression of the acute epilepsy phase and PEA&#39;s potential to alleviate it.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12(S)-HETE</td>
			<td>Biomol</td>
			<td>Cay10007248-25</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>12(S)-HETE-d8</td>
			<td>Biomol</td>
			<td>Cay334570-25</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>1200 series LC System</td>
			<td>Agilent</td>
			<td></td>
			<td>Instrumentation/LCMS</td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td></td>
			<td>Instrumentation/qPCR</td>
		</tr>
		<tr>
			<td>5(S)-HETE-d8</td>
			<td>Biomol</td>
			<td>Cay 334230</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>ABI 7300 Real-Time PCR cycler</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>Instrumentation/qPCR</td>
		</tr>
		<tr>
			<td>Acetonitrile LC-MS Chroma Solv</td>
			<td>Honeywell</td>
			<td>9814920</td>
			<td>Solvent/LCMS</td>
		</tr>
		<tr>
			<td>amber eppendorf tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>Analyst 1.6.2 Software</td>
			<td>AB SCIEX, Darmstadt</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Mettler Toledo</td>
			<td></td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Arachidonic Acid-d8 MS Standard</td>
			<td>Biomol</td>
			<td>Cay-10007277</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Bessmann Tissue Pulverizer</td>
			<td>Spectrum Laboratories, Inc. (Breda, Netherlands)</td>
			<td></td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Bino</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>cleaved Caspase 3 antibody</td>
			<td>Cellsignaling</td>
			<td>9661S</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Cryostat, Leica CM3050 S</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>CTC HTC PAL autosampler</td>
			<td>CTC Analytics AG</td>
			<td></td>
			<td>Instrumentation/LCMS</td>
		</tr>
		<tr>
			<td>Dumont Curved Forceps Dumoxel #7</td>
			<td>FST</td>
			<td>11271-30</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Dumont Forceps Super fine tip #5SF (x2)</td>
			<td>FST</td>
			<td>11252-00</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>EDTA 1000 A R&#246;hrchen</td>
			<td>Kabe Labortechnik</td>
			<td>078001</td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>EP-1 EconoPump</td>
			<td>BioRAD</td>
			<td>700BR07757</td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Fine Forceps Mirror Finish</td>
			<td>FST</td>
			<td>11412-11</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Fine Iris Scissors straight sharp</td>
			<td>FST</td>
			<td>14094-11</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Fine Scissor Tungsten Carbide straight</td>
			<td>FST</td>
			<td>14568-09</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Iris Spatulae</td>
			<td>FST</td>
			<td>10094-13</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Kainic acid</td>
			<td>Abcam</td>
			<td>ab120100</td>
			<td>Epileptic drug</td>
		</tr>
		<tr>
			<td>Lipid View software</td>
			<td>AB SCIEX, Darmstadt</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>LPC 17:0</td>
			<td>Avanis Polaris</td>
			<td>855676P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>LPC 18:0</td>
			<td>Avanis Polaris</td>
			<td>855775P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Luna 2,5&#181;m C18(2)- HAST 100A LC column</td>
			<td>Phenomenex</td>
			<td>00D-4446-B0</td>
			<td>Instrumentation/LCMS</td>
		</tr>
		<tr>
			<td>Magnifying lamp</td>
			<td>Maul GmbH</td>
			<td></td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Methanol LC-MS Chroma Solv 99.9%</td>
			<td>Honeywell</td>
			<td>9814920</td>
			<td>Solvent/LCMS</td>
		</tr>
		<tr>
			<td>Motic Camara</td>
			<td>Motic</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>MTBE</td>
			<td>Honeywell</td>
			<td>34875-1L</td>
			<td>Solvent/LCMS</td>
		</tr>
		<tr>
			<td>MultiQuant 3.0 quantitation software package</td>
			<td>AB SCIEX, Darmstadt</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>NanoDrop 2000c Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Instrumentation/qPCR</td>
		</tr>
		<tr>
			<td>PA 16:0-18:1</td>
			<td>Avanis Polaris</td>
			<td>840857P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PA 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1404</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Palmitoyl Ethanolamide</td>
			<td>Biomol</td>
			<td>Cay90350-100</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Palmitoyl Ethanolamide-d5</td>
			<td>Biomol</td>
			<td>Cay9000573-5</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PC 16:0-18:1</td>
			<td>Avanis Polaris</td>
			<td>850457P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PC 16:0-18:1</td>
			<td>Avanis Polaris</td>
			<td>850457P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PC 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1004</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PE 16:0-18:1</td>
			<td>Avanis Polaris</td>
			<td>850757P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PE 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1104</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PG 16:0-18:1</td>
			<td>Avanis Polaris</td>
			<td>840457P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PG 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1204</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PI 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1504</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Precelleys 24</td>
			<td>Peqlab</td>
			<td></td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Precellys Keramik-K&#252;gelchen</td>
			<td>Peqlab</td>
			<td>91-pcs-ck14p</td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>Precellys Stahlkugeln 2,8mm</td>
			<td>Peqlab</td>
			<td>91-PCS-MK28P</td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>Precellys-keramik-kit 1,4 mm</td>
			<td>VWR</td>
			<td>91-PCS-CK14</td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>Prostaglandin D2</td>
			<td>Biomol</td>
			<td>Cay 12010</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Prostaglandin D2-d4</td>
			<td>Biomol</td>
			<td>Cay 312010</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Prostaglandin E2</td>
			<td>Biomol</td>
			<td>Cay10007211-1</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Prostaglandin E2-d9</td>
			<td>Biomol</td>
			<td>Cay10581-50</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>PS 17:0-14:1</td>
			<td>Avanis Polaris</td>
			<td>LM-1304</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Q Trap 5500 triple-quadrupole linear ion trap MS</td>
			<td>AB SCIEX</td>
			<td>AU111609004</td>
			<td>Instrumentation/LCMS</td>
		</tr>
		<tr>
			<td>Real Time PCR System</td>
			<td>Appliert Biosystem</td>
			<td></td>
			<td>Instrumentation/qPCR</td>
		</tr>
		<tr>
			<td>Resolvin D1</td>
			<td>Biomol</td>
			<td>Cay10012554-11</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Rneasy Mini Kit - RNAase-Free DNase Set (50)</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td>Sample Prep.</td>
		</tr>
		<tr>
			<td>Security Guard precolumn</td>
			<td>Phenomenex</td>
			<td></td>
			<td>Instrumentation/LCMS</td>
		</tr>
		<tr>
			<td>Shandon coverplates</td>
			<td>Thermo Fisher</td>
			<td>72110017</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Shandon slide rack and lid</td>
			<td>Thermo Fisher</td>
			<td>73310017</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>SM 18:0</td>
			<td>Avanis Polaris</td>
			<td>860586P</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>SM d18:1/12:0</td>
			<td>Avanis Polaris</td>
			<td>LM-2312</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Standard Forceps straight Smooth</td>
			<td>FST</td>
			<td>11016-17</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>Surgical Scissor ToughCut Standard Pattern</td>
			<td>FST</td>
			<td>14130-17</td>
			<td>Surgical Tools</td>
		</tr>
		<tr>
			<td>T3000 Thermocycler</td>
			<td>Biometra</td>
			<td></td>
			<td>Instrumentation/qPCR</td>
		</tr>
		<tr>
			<td>Thromboxane B2</td>
			<td>Biomol</td>
			<td>Cay19030-5</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Thromboxane B2-d4</td>
			<td>Biomol</td>
			<td>Cay319030-25</td>
			<td>Lipid Std</td>
		</tr>
		<tr>
			<td>Tissue Lyser II</td>
			<td>Qiagen/ Retsch</td>
			<td>12120240804</td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Tissue Tek</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Toluidinblau</td>
			<td>Roth</td>
			<td>0300.2</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Vapotherm</td>
			<td>Barkey</td>
			<td>4004734</td>
			<td>Instrumentation/Sample prep.</td>
		</tr>
		<tr>
			<td>Wasser LC-MS Chroma Solv</td>
			<td>VWR</td>
			<td>9814920</td>
			<td>Solvent/LCMS</td>
		</tr>
	</tbody>
</table>

lipidomics and transcriptomics in neurological diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

The set of described protocols may be combined on different levels in an aim-specific fashion, such as choice of animal model, route of sampling, method of extraction and profiling (Figure 1).
In order to determine lipid level changes in the brain and periphery over a time course of an acute epileptic seizure state and to unravel the potential antiepileptic effect13 of PEA an...

Watch this Scientific Journal Video about Lipidomics and Transcriptomics in Neurological Diseases at JoVE.com

Lipidomics and Transcriptomics in Neurological Diseases

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

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.

lipidomics-and-transcriptomics-in-neurological-diseases

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...