Long-Term Treatment with Cannabidiol-Enriched Cannabis Extract Induces Synaptic Changes in the Adolescent Rat Hippocampus.

The endocannabinoid system (eCS) is widely distributed in mammalian tissues and it is classically formed by cannabinoid receptors, endogenous bioactive lipids and its synthesis and degradation enzymes. Due to the modulatory role of eCS in synaptic activity in the Central Nervous System (CNS), phytocannabinoids have been increasingly used for the treatment of neurological disorders, even though little is known in terms of the long-term effect of these treatments on CNS development, mainly in the timeframe that comprises childhood and adolescence. Furthermore, an increased number of clinical trials using full-spectrum Cannabis extracts has been seen, rather than the isolated form of phytocannabinoids, when exploring the therapeutical benefits of the Cannabis plant. Thus, this study aims to evaluate the effect of cannabidiol (CBD)-enriched Cannabis extract on synaptic components in the hippocampus of rats from adolescence to early adulthood (postnatal day 45 to 60). Oral treatment of healthy male Wistar rats with a CBD-enriched Cannabis extract (3 mg/kg/day CBD) during 15 days did not affect food intake and water balance. There was also no negative impact on locomotor behaviour and cognitive performance. However, the hippocampal protein levels of GluA1 and GFAP were reduced in animals treated with the extract, whilst PSD95 levels were increased, which suggests rearrangement of glutamatergic synapses and modulation of astrocytic features. Microglial complexity was reduced in CA1 and CA3 regions, but no alterations in their phagocytic activity have been identified by Iba-1 and LAMP2 co-localization. Collectively, our data suggest that CBD-enriched Cannabis treatment may be safe and well-tolerated in healthy subjects, besides acting as a neuroprotective agent against hippocampal alterations related to the pathogenesis of excitatory and astrogliosis-mediated disorders in CNS.

Activation of cannabinoid type 1 receptor (CB1) modulates oligodendroglial process branching complexity in rat hippocampal cultures stimulated by olfactory ensheathing glia-conditioned medium.

The endocannabinoid system (ECS) refers to a complex cell-signaling system highly conserved among species formed by numerous receptors, lipid mediators (endocannabinoids) and synthetic and degradative enzymes. It is widely distributed throughout the body including the CNS, where it participates in synaptic signaling, plasticity and neurodevelopment. Besides, the olfactory ensheathing glia (OEG) present in the olfactory system is also known to play an important role in the promotion of axonal growth and/or myelination. Therefore, both OEG and the ECS promote neurogenesis and oligodendrogenesis in the CNS. Here, we investigated if the ECS is expressed in cultured OEG, by assessing the main markers of the ECS through immunofluorescence, western blotting and qRT-PCR and quantifying the content of endocannabinoids in the conditioned medium of these cells. After that, we investigated whether the production and release of endocannabinoids regulate the differentiation of oligodendrocytes co-cultured with hippocampal neurons, through Sholl analysis in oligodendrocytes expressing O4 and MBP markers. Additionally, we evaluated through western blotting the modulation of downstream pathways such as PI3K/Akt/mTOR and ERK/MAPK, being known to be involved in the proliferation and differentiation of oligodendrocytes and activated by CB1, which is the major endocannabinoid responsive receptor in the brain. Our data show that OEG expresses key genes of the ECS, including the CB1 receptor, FAAH and MAGL. Besides, we were able to identify AEA, 2-AG and AEA related mediators palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in the conditioned medium of OEG cultures. These cultures were also treated with URB597 10-9 M, a FAAH selective inhibitor, or JZL184 10-9 M, a MAGL selective inhibitor, which led to the increase in the concentrations of OEA and 2-AG in the conditioned medium. Moreover, we found that the addition of OEG conditioned medium (OEGCM) enhanced the complexity of oligodendrocyte process branching in hippocampal mixed cell cultures and that this effect was inhibited by AM251 10-6 M, a CB1 receptor antagonist. However, treatment with the conditioned medium enriched with OEA or 2-AG did not alter the process branching complexity of premyelinating oligodendrocytes, while decreased the branching complexity in mature oligodendrocytes. We also observed no change in the phosphorylation of Akt and ERK 44/42 in any of the conditions used. In conclusion, our data show that the ECS modulates the number and maturation of oligodendrocytes in hippocampal mixed cell cultures.

Phytocannabinoids and Cannabis-Based Products as Alternative Pharmacotherapy in Neurodegenerative Diseases: From Hypothesis to Clinical Practice.

Historically, Cannabis is one of the first plants to be domesticated and used in medicine, though only in the last years the amount of Cannabis-based products or medicines has increased worldwide. Previous preclinical studies and few published clinical trials have demonstrated the efficacy and safety of Cannabis-based medicines in humans. Indeed, Cannabis-related medicines are used to treat multiple pathological conditions, including neurodegenerative disorders. In clinical practice, Cannabis products have already been introduced to treatment regimens of Alzheimer's disease, Parkinson's disease and Multiple Sclerosis's patients, and the mechanisms of action behind the reported improvement in the clinical outcome and disease progression are associated with their anti-inflammatory, immunosuppressive, antioxidant, and neuroprotective properties, due to the modulation of the endocannabinoid system. In this review, we describe the role played by the endocannabinoid system in the physiopathology of Alzheimer, Parkinson, and Multiple Sclerosis, mainly at the neuroimmunological level. We also discuss the evidence for the correlation between phytocannabinoids and their therapeutic effects in these disorders, thus describing the main clinical studies carried out so far on the therapeutic performance of Cannabis-based medicines.

Cannabinoid Therapeutics in Chronic Neuropathic Pain: From Animal Research to Human Treatment.

Despite the importance of pain as a warning physiological system, chronic neuropathic pain is frequently caused by damage in the nervous system, followed by persistence over a long period, even in the absence of dangerous stimuli or after healing of injuries. Chronic neuropathic pain affects hundreds of millions of adults worldwide, creating a direct impact on quality of life. This pathology has been extensively characterized concerning its cellular and molecular mechanisms, and the endocannabinoid system (eCS) is widely recognized as pivotal in the development of chronic neuropathic pain. Scientific evidence has supported that phyto-, synthetic and endocannabinoids are efficient for pain management, while strong data arise from the therapeutic use of Cannabis-derived products. The use of medicinal Cannabis products is directed toward not only relieving symptoms of chronic pain, but also improving several aspects of patients' welfare. Here, we review the involvement of eCS, along with other cellular and molecular elements, in chronic neuropathic pain pathology and how this system can be targeted for pain management.

Experimental ischemia/reperfusion model impairs endocannabinoid signaling and Na+/K+ ATPase expression and activity in kidney proximal tubule cells.

LLC-PK1 cells, an immortalized epithelial cell line derived from pig renal proximal tubules, express all the major players of the endocannabinoid system (ECS) such as CB1, CB2 and TRPV1 receptors, as well as the main enzymes involved in the biosynthesis and degradation of the major endocannabinoids named 2-arachidonoylglycerol, 2-AG and anandamide, AEA. Here we investigated whether the damages caused by ischemic insults either in vitro using LLC-PK1 cells exposed to antimycin A (an inductor of ATP-depletion) or in vivo using Wistar rats in a classic renal ischemia and reperfusion (IR) protocol, lead to changes in AEA and 2-AG levels, as well as altered expression of genes from the main enzymes involved in the regulation of the ECS. Our data show that the mRNA levels of the CB1 receptor gene were downregulated, while the transcript levels of monoacylglycerol lipase (MAGL), the main 2-AG degradative enzyme, were upregulated in LLC-PK1 cells after IR model. Accordingly, IR was accompanied by a significant reduction in the levels of 2-AG and AEA, as well as of the two endocannabinoid related molecules, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in LLC-PK1 cells. In kidney cortex homogenates, only AEA levels were significantly decreased. In addition, we found that in both the in vitro and in vivo model IR caused a reduction in the expression and activity of the Na+/K+ ATPase. These changes were reversed by the CB1/CB2 agonist WIN55,212, in a CB1-receptor dependent manner in the LLC-PK1 IR model. In conclusion, the ECS and Na+/K+ ATPase are down-regulated following IR in LLC-PK1 cells and rat kidney. We suggest that CB1 agonists might represent a potential strategy to reverse the consequences of IR injury in kidney tissues.

Bioactive lipids are altered in the kidney of chronic undernourished rats: is there any correlation with the progression of prevalent nephropathies?

BACKGROUND: Undernutrition during childhood leads to chronic diseases in adult life including hypertension, diabetes and chronic kidney disease. Here we explore the hypothesis that physiological alterations in the bioactive lipids pattern within kidney tissue might be involved in the progression of chronic kidney disease. METHODS: Membrane fractions from kidney homogenates of undernourished rats (RBD) were submitted to lipid extraction and analysis by thin layer chromatography and cholesterol determination. RESULTS: Kidneys from RBD rats had 25% lower cholesterol content, which disturb membrane microdomains, affecting Ca2+ homeostasis and the enzymes responsible for important lipid mediators such as phosphatidylinositol-4 kinase, sphingosine kinase, diacylglicerol kinase and phospholipase A2. We observed a decrease in phosphatidylinositol(4)-phosphate (8.8 ± 0.9 vs. 3.6 ± 0.7 pmol.mg-1.mim-1), and an increase in phosphatidic acid (2.2 ± 0.8 vs. 3.8 ± 1.3 pmol.mg-1.mim-1), being these lipid mediators involved in the regulation of key renal functions. Ceramide levels are augmented in kidney tissue from RBD rats (18.7 ± 1.4 vs. 21.7 ± 1.5 fmol.mg-1.min-1) indicating an ongoing renal lesion. CONCLUSION: Results point to an imbalance in the bioactive lipid generation with further consequences to key events related to kidney function, thus contributing to the establishment of chronic kidney disease.

Mechanisms involving Ang II and MAPK/ERK1/2 signaling pathways underlie cardiac and renal alterations during chronic undernutrition.

BACKGROUND: Several studies have correlated protein restriction associated with other nutritional deficiencies with the development of cardiovascular and renal diseases. The driving hypothesis for this study was that Ang II signaling pathways in the heart and kidney are affected by chronic protein, mineral and vitamin restriction. METHODOLOGY/PRINCIPAL FINDINGS: Wistar rats aged 90 days were fed from weaning with either a control or a deficient diet that mimics those used in impoverished regions worldwide. Such restriction simultaneously increased ouabain-insensitive Na+-ATPase and decreased (Na++K+)ATPase activity in the same proportion in cardiomyocytes and proximal tubule cells. Type 1 angiotensin II receptor (AT1R) was downregulated by that restriction in both organs, whereas AT2R decreased only in the kidney. The PKC/PKA ratio increased in both tissues and returned to normal values in rats receiving Losartan daily from weaning. Inhibition of the MAPK pathway restored Na+-ATPase activity in both organs. The undernourished rats presented expanded plasma volume, increased heart rate, cardiac hypertrophy, and elevated systolic pressure, which also returned to control levels with Losartan. Such restriction led to electrical cardiac remodeling represented by prolonged ventricular repolarization parameters, induced triggered activity, early after-depolarization and delayed after-depolarization, which were also prevented by Losartan. CONCLUSION/SIGNIFICANCE: The mechanisms responsible for these alterations are underpinned by an imbalance in the PKC- and PKA-mediated pathways, with participation of angiotensin receptors and by activation of the MAPK/ERK1/2 pathway. These cellular and molecular alterations culminate in cardiac electric remodeling and in the onset of hypertension in adulthood.

Reduced cholesterol levels in renal membranes of undernourished rats may account for urinary Na⁺ loss.

PURPOSE: It has been demonstrated that reabsorption of Na⁺ in the thick ascending limb is reduced and the ability to concentrate urine can be compromised in undernourished individuals. Alterations in phospholipid and cholesterol content in renal membranes, leading to Na⁺ loss and the inability to concentrate urine, were investigated in undernourished rats. METHODS: Sixty-day-old male Wistar rats were utilized to evaluate (1) phospholipid and cholesterol content in the membrane fraction of whole kidneys, (2) cholesterol content and the levels of active Na⁺ transporters, (Na⁺ + K⁺)ATPase and Na⁺-ATPase, in basolateral membranes of kidney proximal tubules, and (3) functional indicators of medullary urine concentration. RESULTS: Body weight in the undernourished group was 73 % lower than in control. Undernourishment did not affect the levels of cholesterol in serum or in renal homogenates. However, membranes of whole kidneys revealed 56 and 66 % reduction in the levels of total phospholipids and cholesterol, respectively. Furthermore, cholesterol and (Na⁺ + K⁺)ATPase activity in proximal tubule membranes were reduced by 55 and 68 %, respectively. Oxidative stress remained unaltered in the kidneys of undernourished rats. In contrast, Na⁺-ATPase activity, an enzyme with all regulatory components in membrane, was increased in the proximal tubules of undernourished rats. Free water clearance and fractional Na⁺ excretion were increased by 86 and 24 %, respectively, and urinary osmolal concentration was 21 % lower in undernourished rats than controls. CONCLUSION: Life-long undernutrition reduces the levels of total phospholipids and cholesterol in membranes of renal tubular cells. This alteration in membrane integrity could diminish (Na⁺ + K⁺)ATPase activity resulting in reduced Na⁺ reabsorption and urinary concentrating ability.