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.

Comparison of effect of crush or transection peripheral nerve lesion on lumbar spinal cord synaptic plasticity and microglial dynamics.

In an injury to the peripheral nervous system, the spinal cord and brain structure reorganize connections to optimize the function of the remaining parts. Many cell events are triggered in the spinal cord to support changes in the synaptic connections around motoneurons, where old connections are removed, and new ones created. Microglial cells are primitive macrophages that invade the central nervous system in early stages of neurodevelopment and have several functions, such as eliminating synapses. We investigated the synaptic plasticity after different types of peripheral (sciatic) nerve injury (crush or total transection), as well as the behavior of microglial cells for 2 weeks after a peripheral lesion. As expected, sciatic-nerve injury reduced motor performance in mice, but crushed animals regained partial motor control. Because of sciatic-nerve injury, pre-synaptic inputs decreased around the motoneurons in the ventro-lateral horn, while microglial cells increased around these cells. Microglial cells also exhibited altered morphology in both types of peripheral lesion, indicating a similar underlying mechanism of plasticity. To investigate the involvement of microglia in this scenario, microglial activation was modulated by daily administration of minocycline. The minocycline treatment directly affected the microglial response and impacted the synapse rearrangement in the spinal cord. Together, these results demonstrate that microglia cells are involved in synaptic plasticity in the lumbar spinal cord in both nerve-injury scenarios. SUMMARY OF STATEMENT: Here, we demonstrated that acute plasticity in the lumbar spinal cord (LSC) did not differ between crush and transection of peripheral nerve, and that microglial reactivity in the LSC was important after both injury types.

CXCR4 and MIF are required for neutrophil extracellular trap release triggered by Plasmodium-infected erythrocytes.

Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.

Mesenchymal Stromal Cells Protect the Blood-Brain Barrier, Reduce Astrogliosis, and Prevent Cognitive and Behavioral Alterations in Surviving Septic Mice.

OBJECTIVES: Survivors of sepsis are frequently left with significant cognitive and behavioral impairments. These complications derive from nonresolving inflammation that persists following hospital discharge. To date, no study has investigated the effects of mesenchymal stromal cell therapy on the blood-brain barrier, astrocyte activation, neuroinflammation, and cognitive and behavioral alterations in experimental sepsis. DESIGN: Prospective, randomized, controlled experimental study. SETTING: Government-affiliated research laboratory. SUBJECTS: Male Swiss Webster mice (n = 309). INTERVENTIONS: Sepsis was induced by cecal ligation and puncture; sham-operated animals were used as control. All animals received volume resuscitation (1 mL saline/mouse subcutaneously) and antibiotics (meropenem 10 mg/kg intraperitoneally at 6, 24, and 48 hours). Six hours after surgery, mice were treated with mesenchymal stromal cells IV (1 × 10 cells in 0.05 mL of saline/mouse) or saline (0.05 mL IV). MEASUREMENTS AND MAIN RESULTS: At day 1, clinical score and plasma levels of inflammatory mediators were increased in cecal ligation and puncture mice. Mesenchymal stromal cells did not alter clinical score or survival rate, but reduced levels of systemic interleukin-1ß, interleukin-6, and monocyte chemoattractant protein-1. At day 15, survivor mice completed a battery of cognitive and behavioral tasks. Cecal ligation and puncture mice exhibited spatial and aversive memory deficits and anxiety-like behavior. These effects may be related to increased blood-brain barrier permeability, with altered tight-junction messenger RNA expression, increased brain levels of inflammatory mediators, and astrogliosis (induced at day 3). Mesenchymal stromal cells mitigated these cognitive and behavioral alterations, as well as reduced blood-brain barrier dysfunction, astrocyte activation, and interleukin-1ß, interleukin-6, tumor necrosis factor-α, and interleukin-10 levels in vivo. In cultured primary astrocytes stimulated with lipopolysaccharide, conditioned media from mesenchymal stromal cells reduced astrogliosis, interleukin-1ß, and monocyte chemoattractant protein-1, suggesting a paracrine mechanism of action. CONCLUSIONS: In mice who survived experimental sepsis, mesenchymal stromal cell therapy protected blood-brain barrier integrity, reduced astrogliosis and neuroinflammation, as well as improved cognition and behavior.