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      Anti-apoptotic and Immunomodulatory Effect of CB2 Agonist, JWH133, in a Neonatal Rat Model of Hypoxic-Ischemic Encephalopathy

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          Introduction: Neonatal HIE is associated with high morbidity and mortality. Current research, is focused on developing alternative treatments to therapeutic hypothermia for treatment of HIE. The endocannabinoid system is known to be influential in neuronal protection. Activation of brain CB2 receptors, has been shown to reduce inflammatory markers and decrease infarct volume in adult cerebral ischemic models.

          Methods: Rat pups were divided into six groups: 1—Placebo; 2—JWH133; 3—HIE + Placebo; 4—HIE + JWH133; 5—HIE + Hypothermia + Placebo; and 6—HIE + Hypothermia + JWH133. HIE was induced in in groups 3–6 by right carotid ligation on postnatal day 7 followed by placement in a hypoxic chamber. Pups in groups 5 and 6 were treated with hypothermia. Western blot analysis was used to analyze brain tissue for acute inflammatory markers (IL-6, TNFα, MIP1α, and RANTES), immunoregulatory cytokines (TGFβ and IL-10), and CB2 receptor expression. DNA fragmentation in the brains of pups was determined via TUNEL staining post HIE.

          Results: The combination of JWH133 and hypothermia significantly reduced tumor necrosis factor α (TNFα) (−57.7%, P = 0.0072) and macrophage inflammatory protein 1α (MIP1α) (−50.0%, P = 0.0211) as compared to placebo. DNA fragmentation was also significantly reduced, with 6.9 ± 1.4% TUNEL+ cells in HIE+JWH133 and 12.9 ± 2.2% in HIE+Hypothermia + JWH133 vs. 16.6 ± 1.9% in HIE alone. No significant difference was noted between groups for the expression of interleukins 6 and 10, RANTES, or TGFβ. After 8 h, CB2 receptor expression increased nearly 2-fold in the HIE and HIE + JWH133 groups (+214%, P = 0.0102 and +198%, P = 0.0209, respectively) over placebo with no significant change in the hypothermia groups. By 24 h post HIE, CB2 receptor expression was elevated over five times that of placebo in the HIE ( P < 0.0001) and HIE + JWH133 ( P = 0.0002) groups, whereas hypothermia treatment maintained expression similar to that of placebo animals.

          Conclusion: These results indicate that the combination of CB2 agonist and hypothermia may be neuroprotective in treating HIE, opening the door for further studies to examine alternative or adjuvant therapies to hypothermia.

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          Most cited references 18

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          Molecular characterization of a peripheral receptor for cannabinoids.

          The major active ingredient of marijuana, delta 9-tetrahydrocannabinol (delta 9-THC), has been used as a psychoactive agent for thousands of years. Marijuana, and delta 9-THC, also exert a wide range of other effects including analgesia, anti-inflammation, immunosuppression, anticonvulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting. The clinical application of cannabinoids has, however, been limited by their psychoactive effects, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of delta 9-THC, and more recently from the cloning of a gene encoding a G-protein-coupled receptor for cannabinoids. This receptor is expressed in the brain but not in the periphery, except for a low level in testes. It has been proposed that the nonpsychoactive effects of cannabinoids are either mediated centrally or through direct interaction with other, non-receptor proteins. Here we report the cloning of a receptor for cannabinoids that is not expressed in the brain but rather in macrophages in the marginal zone of spleen.
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            Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations.

            Two proteins with seven transmembrane-spanning domains typical of guanosine-nucleotide-binding-protein-coupled receptors have been identified as cannabinoid receptors; the central cannabinoid receptor, CB1, and the peripheral cannabinoid receptor, CB2, initially described in rat brain and spleen, respectively. Here, we report the distribution patterns for both CB1 and CB2 transcripts in human immune cells and in several human tissues, as analysed using a highly sensitive and quantitative PCR-based method. CB1 was mainly expressed in the central nervous system and, to a lower extent, in several peripheral tissues such as adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils. In contrast, the CB2 gene, which is not expressed in the brain, was particularly abundant in immune tissues, with an expression level 10-100-fold higher than that of CB1. Although CB2 mRNA was also detected in some other peripheral tissues, its level remained very low. In spleen and tonsils, the CB2 mRNA content was equivalent to that of CB1 mRNA in the central nervous system. Among the main human blood cell subpopulations, the distribution pattern of the CB2 mRNA displayed important variations. The rank order of CB2 mRNA levels in these cells was B-cells > natural killer cells > monocytes > polymorphonuclear neutrophil cells > T8 cells > T4 cells. The same rank order was also established in human cell lines belonging to the myeloid, monocytic and lymphoid lineages. The prevailing expression of the CB2 gene in immune tissues was confirmed by Northern-blot analysis. In addition, the expression of the CB2 protein was demonstrated by an immunohistological analysis performed on tonsil sections using specific anti-(human CB2) IgG; this experiment showed that CB2 expression was restricted to B-lymphocyte-enriched areas of the mantle of secondary lymphoid follicles. These results suggest that (a) CB1 and CB2 can be considered as tissue-selective antigens of the central nervous system and immune system, respectively, and (b) cannabinoids may exert specific receptor-mediated actions on the immune system through the CB2 receptor.
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              Modulation of the cannabinoid CB2 receptor in microglial cells in response to inflammatory stimuli.

              The cannabinoid system is known to be important in neuronal regulation, but is also capable of modulating immune function. Although the CNS resident microglial cells have been shown to express the CB2 subtype of cannabinoid receptor during non-immune-mediated pathological conditions, little is known about the expression of the cannabinoid system during immune-mediated CNS pathology. To examine this question, we measured CB2 receptor mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) and, by real-time PCR, found a 100-fold increase in CB2 receptor mRNA expression during EAE onset. We next determined whether microglial cells specifically express the CB2 receptor during EAE, and found that activated microglial cells expressed 10-fold more CB2 receptor than microglia in the resting state. To determine the signals required for the up-regulation of the CB2 receptor, we cultured microglial cells with combinations of gamma-interferon (IFN-gamma) and granulocyte) macrophage-colony stimulating factor (GM-CSF), which both promote microglial cell activation and are expressed in the CNS during EAE, and found that they synergized, resulting in an eight to 10-fold increase in the CB2 receptor. We found no difference in the amount of the CB2 receptor ligand, 2-arachidonylglycerol (2-AG), in the spinal cord during EAE. These data demonstrate that microglial cell activation is accompanied by CB2 receptor up-regulation, suggesting that this receptor plays an important role in microglial cell function in the CNS during autoimmune-induced inflammation.

                Author and article information

                Front Pediatr
                Front Pediatr
                Front. Pediatr.
                Frontiers in Pediatrics
                Frontiers Media S.A.
                27 February 2020
                : 8
                1Division of Neonatology, Department of Pediatrics, Advocate Children's Hospital , Park Ridge, IL, United States
                2Chicago College of Pharmacy, Midwestern University , Downers Grove, IL, United States
                3Advocate Center for Pediatric Research, Advocate Children's Hospital , Park Ridge, IL, United States
                Author notes

                Edited by: Henry J. Rozycki, Virginia Commonwealth University, United States

                Reviewed by: Bruce David Uhal, Michigan State University, United States; Surendra Sharma, Women & Infants Hospital of Rhode Island, United States

                *Correspondence: Bhavna Gupta bhavna.0388@ 123456gmail.com

                This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics

                †These authors have contributed equally to this work

                Copyright © 2020 Gupta, Hornick, Briyal, Donovan, Prazad and Gulati.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 6, Tables: 0, Equations: 0, References: 31, Pages: 9, Words: 5495
                Original Research


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