Release of Endocannabinoids into the Cerebrospinal Fluid during the Induction of the Trigemino-Hypoglossal Reflex in Rats
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals and Anaesthesia
2.2. Chemicals
2.3. Perfusion of Cerebral Ventricles in Rats
2.4. Tooth Pulp Stimulation
2.5. Recording Tongue Jerks
2.6. Experimental Design
2.7. ELISA Test
2.8. RNA Isolation and Gene Expression Analysis
2.8.1. Total RNA Isolation
2.8.2. Quality Analysis of Isolated RNA
2.8.3. RT-PCR Reverse Transcription
2.8.4. Real-Time PCR Reaction
2.9. Statistical Analysis
3. Results
3.1. Effect of Tooth Pulp Stimulation and URB597 Treatment on the AEA and 2-AG Concentration in the CSF
3.2. Correlations between AEA and 2-AG Concentrations in CSF and CB1R and CB2R Expression in the Rat Brain Structures
4. Discussion
Limitations
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
2-AG | 2-arachidonoylglycerol |
aCSF | artificial cerebrospinal fluid |
AEA | N-arachidonyl ethanol amine, anandamide |
BBB | blood–brain barrier |
CB | cannabinoid |
CB1R | cannabinoid receptor type 1 |
CB2R | cannabinoid receptor type 2 |
CBR | cannabinoid receptor |
CNS | central nervous system |
CSF | cerebrospinal fluid |
DAGL | diacylglycerol lipase |
DMSO | dimethyl sulfoxide |
EC | endocannabinoid |
ECS | endocannabinoid system |
ETJ | evoked tongue jerks |
FAAH | fatty acid amide hydrolase |
MAGL | monoacylglycerol lipase |
NTC | nucleus trigeminalis caudalis |
PAG | periaqueductal central gray |
RVM | rostral ventromedial medulla |
URB597 | FAAH inhibitor-degrading enzyme of AEA |
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Gene | Forward Primers | Reverse Primers |
---|---|---|
GAPDH | 5′-GTCGCTGTTGAAGTCAGAGGAG-3′ | 5′-CGTGTCAGTGGTGGACCTGAC-3′ |
CB1R | 5′-CTACTGGTGCTGTGTGTCATC-3′ | 5′-GCTGTCTTTACGGTGGAATAC-3′ |
CB2R | 5′-AGGTTGCATTCCCAACAGAC-3′ | 5′-TTAGTTCCTCTGGGCAATGG-3′ |
Endocannabinoids | Relative Quantity of CB1R mRNA—Mesencephalon | Relative Quantity of CB1R mRNA—Thalamus | Relative Quantity of CB1R mRNA—Hypothalamus | Relative Quantity of CB2R mRNA—Mesencephalon | Relative Quantity of CB2R mRNA—Thalamus | Relative Quantity of CB2R mRNA—Hypothalamus |
---|---|---|---|---|---|---|
AEA CSF [ng/mL] | 0.60 p = 0.008 | 0.26 p = 0.292 | 0.62 p = 0.006 | −0.14 p = 0.587 | −0.59 p = 0.011 | −0.21 p = 0.398 |
2-AG CSF [ng/mL] | −0.51 p = 0.032 | −0.41 p = 0.093 | −0.28 p = 0.267 | 0.48 p = 0.042 | 0.32 p = 0.193 | 0.40 p = 0.104 |
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Zubrzycki, M.; Zubrzycka, M.; Wysiadecki, G.; Szemraj, J.; Jerczynska, H.; Stasiolek, M. Release of Endocannabinoids into the Cerebrospinal Fluid during the Induction of the Trigemino-Hypoglossal Reflex in Rats. Curr. Issues Mol. Biol. 2022, 44, 2401-2416. https://doi.org/10.3390/cimb44050164
Zubrzycki M, Zubrzycka M, Wysiadecki G, Szemraj J, Jerczynska H, Stasiolek M. Release of Endocannabinoids into the Cerebrospinal Fluid during the Induction of the Trigemino-Hypoglossal Reflex in Rats. Current Issues in Molecular Biology. 2022; 44(5):2401-2416. https://doi.org/10.3390/cimb44050164
Chicago/Turabian StyleZubrzycki, Marek, Maria Zubrzycka, Grzegorz Wysiadecki, Janusz Szemraj, Hanna Jerczynska, and Mariusz Stasiolek. 2022. "Release of Endocannabinoids into the Cerebrospinal Fluid during the Induction of the Trigemino-Hypoglossal Reflex in Rats" Current Issues in Molecular Biology 44, no. 5: 2401-2416. https://doi.org/10.3390/cimb44050164