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Research Article
Aidan Levine
University of Arizona Arizona Health Sciences Center: The University of Arizona Health Sciences
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1808-5228
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Several chronic pain disorders, such as migraine and fibromyalgia, have an increased prevalence in the female population. The underlying mechanisms of this sex-biased prevalence have yet to be thoroughly documented but could be related to endogenous differences in neuromodulators in pain networks, including the endocannabinoid system. The cellular endocannabinoid system is comprised of the endogenous lipid signals 2-AG (2-arachidonoylglycerol) and AEA (anandamide); the enzymes that synthesize and degrade them; and the cannabinoid receptors. The relative prevalence of different components of the endocannabinoid system in specific brain regions may alter responses to endogenous and exogenous ligands.
Methods: Brain tissue from naïve male and female Sprague Dawley rats was harvested from V1M cortex, periaqueductal gray, trigeminal nerve, and trigeminal nucleus caudalis. Tissue was analyzed for relative levels of endocannabinoid enzymes, ligands, and receptors via mass spectrometry, unbiased proteomic analysis, and immunohistochemistry.
Results: Mass spectrometry revealed cortical AEA levels were significantly higher in males compared to females (p<0.001), whereas 2-AG levels in periaqueductal grey were significantly higher in females compared to males (p<0.0001). Immunohistochemistry followed by unbiased proteomics confirmed the prevalence of 2-AG-endocannabinoid system enzymes in the female PAG.
Conclusions: Our results suggest that sex differences exist in the endocannabinoid system in two CNS regions relevant to cortical spreading depression (V1M cortex) and descending modulatory networks in pain/anxiety (PAG). These basal differences in endogenous endocannabinoid mechanisms may facilitate the development of chronic pain conditions and may also underlie sex differences in response to therapeutic intervention.
Keywords
sex differences, endocannabinoids, 2-arachidonoylglycerol, anandamide, migraine, pain, proteomics, periaqueductal grey
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This is a list of supplementary files associated with this preprint. Click to download.
- AdditionalFigure1.jpg
Additional Figure 1: Regional Analysis (A) Serial sections from rats’ brains were obtained at a thickness of 30um, moving posteriorly from approximately Bregma -7mm with PAG imaging in the lateral PAG. (B) Quantification of the relative area immunoreactive in each field for Nissl (neuronal soma), GFAP (astrocytes), and Iba1 (microglia) was statistically similar between regions and sex.
- eCBsexdiffAddlTables110.xlsx
Additional Table 1. Proteins more prevalent in female PAG. (.xlsx) A total of 3396 proteins were more prevalent in female PAG. Of the 3396 proteins with female prevalence, 512 were present at statistically significantly higher levels than in males (highlighted in grey), representing 8.5% of all proteins detected. Additional Table 2. Proteins more prevalent in male PAG. (.xlsx) A total of 2449 proteins were more prevalent in male PAG. In males, 218 proteins were expressed at significantly higher levels as compared to female (3.6% of all proteins detected) (highlighted in grey). Additional Table 3. List of proteins detected in PAG samples without sex-differences. (.xlsx) 164 proteins showed no sex difference in expression. Additional Table 4. Fold Differences (.xlsx) 595 proteins showed a fold difference of 1.2 or greater with more than 75% being detected at higher levels in female PAG. Additional Table 5. GO-MF Female Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in female PAG using the bioinformatic database DAVID was performed for GO-Molecular Function (MF). The top 10 category is highlighted. Additional Table 6. GO-BP Female Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in female PAG using the bioinformatic database DAVID was performed for GO-Biological Processes (BP). The top 10 category is highlighted. Additional Table 7. GO-KEGG Female Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in female PAG using the bioinformatic database DAVID was performed for GO-KEGG pathways. The top 10 category is highlighted. Additional Table 8. GO-MF Male Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in male PAG using the bioinformatic database DAVID was performed for GO-Molecular Function (MF). The top 10 category is highlighted. Additional Table 9.GO-BP Male Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in male PAG using the bioinformatic database DAVID was performed for GO-Biological Processes (BP). The top 10 category is highlighted. Additional Table 10.GO-KEGG Male Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in male PAG using the bioinformatic database DAVID was performed for GO-KEGG pathways. The top 10 category is highlighted.
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Received 10 Jun, 2021
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This preprint is under consideration at Biology of Sex Differences. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Aidan Levine
University of Arizona Arizona Health Sciences Center: The University of Arizona Health Sciences
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1808-5228
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 11 Jun, 2021
No community comments so far
Editorial decision: Major Revision
On 07 Jul, 2021
Review #2 received
Received 06 Jul, 2021
Reviewer #2 agreed
On 28 Jun, 2021
Review #1 received
Received 27 Jun, 2021
Reviews received
Received 10 Jun, 2021
Reviewer #1 agreed
On 10 Jun, 2021
Reviewers invited
Invitations sent on 08 Jun, 2021
Editor assigned
On 07 Jun, 2021
Submission checks complete
On 07 Jun, 2021
Editor invited
On 07 Jun, 2021
First submitted
On 04 Jun, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Several chronic pain disorders, such as migraine and fibromyalgia, have an increased prevalence in the female population. The underlying mechanisms of this sex-biased prevalence have yet to be thoroughly documented but could be related to endogenous differences in neuromodulators in pain networks, including the endocannabinoid system. The cellular endocannabinoid system is comprised of the endogenous lipid signals 2-AG (2-arachidonoylglycerol) and AEA (anandamide); the enzymes that synthesize and degrade them; and the cannabinoid receptors. The relative prevalence of different components of the endocannabinoid system in specific brain regions may alter responses to endogenous and exogenous ligands.
Methods: Brain tissue from naïve male and female Sprague Dawley rats was harvested from V1M cortex, periaqueductal gray, trigeminal nerve, and trigeminal nucleus caudalis. Tissue was analyzed for relative levels of endocannabinoid enzymes, ligands, and receptors via mass spectrometry, unbiased proteomic analysis, and immunohistochemistry.
Results: Mass spectrometry revealed cortical AEA levels were significantly higher in males compared to females (p<0.001), whereas 2-AG levels in periaqueductal grey were significantly higher in females compared to males (p<0.0001). Immunohistochemistry followed by unbiased proteomics confirmed the prevalence of 2-AG-endocannabinoid system enzymes in the female PAG.
Conclusions: Our results suggest that sex differences exist in the endocannabinoid system in two CNS regions relevant to cortical spreading depression (V1M cortex) and descending modulatory networks in pain/anxiety (PAG). These basal differences in endogenous endocannabinoid mechanisms may facilitate the development of chronic pain conditions and may also underlie sex differences in response to therapeutic intervention.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- AdditionalFigure1.jpg
Additional Figure 1: Regional Analysis (A) Serial sections from rats’ brains were obtained at a thickness of 30um, moving posteriorly from approximately Bregma -7mm with PAG imaging in the lateral PAG. (B) Quantification of the relative area immunoreactive in each field for Nissl (neuronal soma), GFAP (astrocytes), and Iba1 (microglia) was statistically similar between regions and sex.
- eCBsexdiffAddlTables110.xlsx
Additional Table 1. Proteins more prevalent in female PAG. (.xlsx) A total of 3396 proteins were more prevalent in female PAG. Of the 3396 proteins with female prevalence, 512 were present at statistically significantly higher levels than in males (highlighted in grey), representing 8.5% of all proteins detected. Additional Table 2. Proteins more prevalent in male PAG. (.xlsx) A total of 2449 proteins were more prevalent in male PAG. In males, 218 proteins were expressed at significantly higher levels as compared to female (3.6% of all proteins detected) (highlighted in grey). Additional Table 3. List of proteins detected in PAG samples without sex-differences. (.xlsx) 164 proteins showed no sex difference in expression. Additional Table 4. Fold Differences (.xlsx) 595 proteins showed a fold difference of 1.2 or greater with more than 75% being detected at higher levels in female PAG. Additional Table 5. GO-MF Female Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in female PAG using the bioinformatic database DAVID was performed for GO-Molecular Function (MF). The top 10 category is highlighted. Additional Table 6. GO-BP Female Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in female PAG using the bioinformatic database DAVID was performed for GO-Biological Processes (BP). The top 10 category is highlighted. Additional Table 7. GO-KEGG Female Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in female PAG using the bioinformatic database DAVID was performed for GO-KEGG pathways. The top 10 category is highlighted. Additional Table 8. GO-MF Male Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in male PAG using the bioinformatic database DAVID was performed for GO-Molecular Function (MF). The top 10 category is highlighted. Additional Table 9.GO-BP Male Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in male PAG using the bioinformatic database DAVID was performed for GO-Biological Processes (BP). The top 10 category is highlighted. Additional Table 10.GO-KEGG Male Analysis (.xlsx) Gene ontology (GO) enrichment analysis of the significantly affected proteins in male PAG using the bioinformatic database DAVID was performed for GO-KEGG pathways. The top 10 category is highlighted.
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