Neuroprotection by the cannabis-related products, cannabidiol and cannabigerol, and their associated mechanisms of action

The discovery and characterization of the endocannabinoid system (ECS) brought out years of research focusing on two aims. The study of its participation in the physiopathology in several diseases, including neurodegenerative disorders, and as a direct or indirect target for treating these disorders by cannabinoids or phytocannabinoids (i.e., specific compounds present in the Cannabis sativa plant). Preclinical evidence and some clinical data have shown the therapeutic potential of the most relevant phytocannabinoids, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), but less for cannabigerol (CBG). In the present review, we summarized data focused on the therapeutic potential of CBD and CBG as neuroprotective agents. This property appears to be exerted by the direct or indirect activation of targets within the ECS and also by mechanisms non-mediated by the ECS. We provide information which could be useful for future CBD and CBG applications in human neurodegenerative diseases treatment.Agencia Nacional de Investigación e Innovació

Impact of different formulations of pharmaceutical cannabis-based extracts on the neuroprotective effect in cerebellar granule cell cultures

Preclinical research supports the benefits of pharmaceutical cannabis-based extracts for treating different medical conditions (e. g., epilepsy); however, their neuroprotective potential has not been widely investigated. In addition, there is still controversy about the impact of other factors in the beneficial effect of these extracts (e.g., the entourage effect, and oil formulations). We evaluated the neuroprotective activity of Epifractan (EPI), a cannabis-based medicinal extract containing a high level of cannabidiol (CBD), components like terpenoids and flavonoids, and trace levels of Δ9-tetrahydrocannabinol and the acid form of CBD. Using primary cultures of cerebellar granule cells, we determined the ability of EPI to counteract the rotenone-induced neurotoxicity by analyzing cell viability and morphology of neurons and astrocytes by immunocytochemical assays. The effect of EPI was compared with XALEX, a plant-derived and highly purified CBD formulation (XAL), and pure CBD crystals (CBD). The results revealed that EPI induced a significant reduction in the rotenone-induced neurotoxicity in a wide range of concentrations without causing neurotoxicity per se. EPI showed a similar effect to XAL suggesting that no additive or synergistic interactions (i.e., entourage effect) between individual substances present in EPI occurred. In contrast, CBD crystals did show a different profile to EPI and XAL since a neurotoxic effect per se was observed at the higher concentrations assayed. Medium-chain triglyceride oil used in EPI formulation could explain this difference. Our data support a neuroprotective effect of EPI which may provide neuroprotection in different neurodegenerative processes. The results highlight the role of CBD as the active component of EPI but also support the need for an appropriate formulation to dilute pharmaceutical cannabis-based productAgencia Nacional de Investigación e Innovació

IMPACT OF DIFFERENT FORMULATIONS OF PHARMACEUTICAL CANNABIS-BASED EXTRACTS ON THE NEUROPROTECTIVE EFFECT IN CEREBELLAR GRANULE CELL CULTURES

Preclinical research supports the benefits of pharmaceutical cannabis-based extracts for treating different medical conditions (e. g., epilepsy); however, their neuroprotective potential has not been widely investigated. In addition, there is still controversy about the impact of other factors in the beneficial effect of these extracts (e.g., the entourage effect, and oil formulations). We evaluated the neuroprotective activity of Epifractan (EPI), a cannabis-based medicinal extract containing a high level of cannabidiol (CBD), components like terpenoids and flavonoids, and trace levels of Δ9- tetrahydrocannabinol and the acid form of CBD. Using primary cultures of cerebellar granule cells, we determined the ability of EPI to counteract the rotenone-induced neurotoxicity by analyzing cell viability and morphology of neurons and astrocytes by immunocytochemical assays. The effect of EPI was compared with XALEX, a plant-derived and highly purified CBD formulation (XAL), and pure CBD crystals (CBD). The results revealed that EPI induced a significant reduction in the rotenone-induced neurotoxicity in a wide range of concentrations without causing neurotoxicity per se. EPI showed a similar effect to XAL suggesting that no additive or synergistic interactions (i.e., entourage effect) between individual substances present in EPI occurred. In contrast, CBD crystals did show a different profile to EPI and XAL since a neurotoxic effect per se was observed at the higher concentrations assayed. Medium-chain triglyceride oil used in EPI formulation could explain this difference. Our data support a neuroprotective effect of EPI which may provide neuroprotection in different neurodegenerative processes. The results highlight the role of CBD as the active component of EPI but also support the need for an appropriate formulation to dilute pharmaceutical cannabis-based productAgencia Nacional de Investigación e Innovació

Neuroprotective effect of a pharmaceutical extract of cannabis with high content on CBD against rotenone in primary cerebellar granule cell cultures and the relevance of formulations

Preclinical research supports the benefits of pharmaceutical cannabis-based extracts for treating different medical conditions (e.g., epilepsy); however, their neuroprotective potential has not been widely investigated. Using primary cultures of cerebellar granule cells, we evaluated the neuroprotective activity of Epifractan (EPI), a cannabis-based medicinal extract containing a high level of cannabidiol (CBD), components like terpenoids and flavonoids, and trace levels of Δ9-tetrahydrocannabinol and the acid form of CBD. We determined the ability of EPI to counteract the rotenone-induced neurotoxicity by analyzing cell viability and morphology of neurons and astrocytes by immunocytochemical assays. The effect of EPI was compared with XALEX, a plant-derived and highly purified CBD formulation (XAL), and pure CBD crystals (CBD). The results revealed that EPI induced a significant reduction in the rotenone induced neurotoxicity in a wide range of concentrations without causing neurotoxicity per se. EPI showed a similar effect to XAL suggesting that no additive or synergistic interactions between individual substances present in EPI occurred. In contrast, CBD did show a different profile to EPI and XAL since a neurotoxic effect per se was observed at the higher concentrations assayed. Medium-chain triglyceride (MCT) oil used in EPI formulation could explain this difference. Our data support a neuroprotective effect of EPI which may provide neuroprotection in different neurodegenerative processes. The results highlight the role of CBD as the active component of EPI but also support the need for an appropriate formulation to dilute pharmaceutical cannabis-based products, which could be critical to avoid neurotoxicity at very high doses.Agencia Nacional de Investigación e Innovació

Uso medicinal de cannabinoides: su acción como agente neuroprotector y su biodisponibilidad cerebral

A pesar de los avances en el conocimiento sobre los cambios fisiopatológicos y moleculares de las diferentes enfermedades neurodegenerativas no existe cura para ninguna de ellas. Las terapias existentes, tanto farmacológicas como quirúrgicas, están destinadas a tratar la sintomatología de estasenfermedades y, en general, poseen importantes efectos adversos. Esto hace imprescindible la necesidad de trabajar en el desarrollo de nuevos tratamientos eficaces. Dada la naturaleza multifactorial de las enfermedades neurodegenerativas, se cree que una combinación de moléculas capaces de actuar simultáneamente sobre varios blancos celulares (acción polifarmacológica) va a tener un mayor potencial neuroprotector en comparación con aquellos fármacos con un solo mecanismo de acción. El proyecto de investigación propone evaluar el potencial neuroprotector de combinaciones de cannabinoides, en diferentes proporciones, usando modelos celulares. Además, busca estudiar algunos mecanismos de acción (dentro y fuera del sistema endocannabinoide) que podrían subyacer a la neuroprotección. Estos estudios permitirán avanzar en la caracterización de los cannabinoides y sus combinaciones para su eventual aplicación terapéutica para enfermedades neurodegenerativasAgencia Nacional de Investigación e Innovació

Increase in perivascular innervation of the human umbilical cord of newborns prenatally exposed to cocaine: impact on clinical variables

Introduction: Substance use during pregnancy represents a critical public health concern, linked with several harmful maternal and fetal consequences. Women are at their highest risk of developing a substance use disorder throughout their reproductive years. Particularly, cocaine use represents a worldwide problem. Given that vasoconstriction is modulated by sympathetic innervation, and cocaine is a sympathomimetic drug, we hypothesized that modifications in this type of innervation around umbilical vessels could compromise maternal filial blood flow, however, the impact of these changes remains to be evaluated. Objectives: Study the perivascular sympathetic innervation in newborns’ umbilical cord (UC) from cocaine pregnant users and seek for correlations between UC innervation and clinical manifestations. The impact of tobacco consumption was also addressed to identify possible deleterious exposure combinations. Methods: Perinatal clinical histories (SIP; by PAHO) of UC donors were evaluated (informed consent: INDT version-N°6/30-10-18). Analyses were conducted in: Control-group (clinically normal pregnancies; no pre-gestational/gestational pathologies); cocaine-group (self reported history of cocaine use during pregnancy); tobacco-group (self-reported history of tobacco smoking without other drug consumption during pregnancy). Influence of polyconsumption, gestational age and mother’s nutritional status were considered. Immunofluorescence: UC cryosections were co-labelled with anti-human PGP 9.5 (Abcam rabbit), a general nerve fiber marker; and anti-TH (Tyrosine Hydroxylase; Millipore-mouse), a specific marker for sympathetic fibers. Results: We found a subpopulation of newborns’ UC from cocaine users that had increased perivascular sympathetic innervation compared to healthy peers. Additionally, there was a negative correlation between the immunoreactive area occupied by nerve fibers in the umbilical arteries and the body weight, size and cephalic perimeter percentiles of newborns. No difference in age, size, weight and BMI (body mass index) of mothers from different groups was found. Also, we confirmed that 66% of UC from newborns of tobacco-group were not innervated. Conclusions: The subpopulation of newborns prenatally exposed to cocaine that had altered innervation in their umbilical arteries were those who presented the lowest size and weight. This supports our hypothesis and reveals a potential mechanism underlying the relationship between developmental disorders and prenatal drug exposure. Our results from the tobacco group will allow us to assess the effect of poly-substance use during pregnancies.Agencia Nacional de Investigación e InnovaciónPrograma de Desarrollo de las Ciencias Básica

Evaluación de la capacidad antioxidante de las variedades de Cannabis Alfa y Beta, comercializadas en farmacias del Uruguay

La ley de regulación del mercado de Cannabis en el Uruguay permitió el acceso a la población de dos variantes de Cannabis, alfa y beta, la primera con predominancia de variedad “índica” y la segunda de “sativa”. Escasos estudios se han realizado hasta ahora sobre los efectos producidos por dichas variedades. Reportes de la literatura muestran que varias de las acciones beneficiosas de extractos de Cannabis se deben a su capacidad antioxidante (CA), la cual podría estar relacionada con el contenido en fenoles totales, que varía según la variedad y el método de extracción. En este trabajo evaluamos la CA de las variedades alfa y beta luego de dos métodos de extracción: 1) EtOH 95% y 3 min de macerado y, 2) EtOH 70% y 10 min de sonicado. Además, evaluamos la CA luego de la descarboxilación por calor de estos extractos, y evaluamos el contenido de fenoles totales, CBD y CBDA. Resultados primarios muestran que el segundo método de extracción fue más eficaz en la extracción de fenoles totales, CBD y CBDA en ambas variedades y, la CA de la variedad beta fue mayor que la alfa. La descarboxilación de los extractos redujo el contenido de fenoles, disminuyó CBDA y aumentó de CBD, y no afectó la CA. En su conjunto, los resultados sugieren que la CA de las variedades no depende únicamente del contenido de fenoles totales, sino también de la forma química en que los compuestos se encuentren, principalmente los cannabinoides (por ejemplo, CBD).Agencia Nacional de Investigación e Innovació

Extracellular matrix stiffness negatively affects axon elongation, growth cone area and F-actin levels in a collagen type I 3D culture

Three dimensional (3D) in vitro neuronal cultures can better reproduce physiologically relevant phenotypes compared to 2D-cultures, because in vivo neurons reside in a 3D microenvironment. Interest in neuronal 3D cultures is emerging, with special attention to the mechanical forces that regulate axon elongation and sprouting in three dimensions. Type I collagen (Col-I) is a native substrate since it is present in the extracellular matrix and hence emulates an in vivo environment to study axon growth. The impact of its mechanical properties needs to be further investigated. Here, we generated Col-I 3D matrices of different mechanical stiffness and evaluated axon growth in three dimensions. Superior cervical ganglion (SCG) explants from neonatal rats were cultured in soft and stiff Col-I 3D matrices and neurite outgrowth was assessed by measuring: maximum neuritic extent; neuritic halo area and fasciculation. Axonal cytoskeletal proteins were examined. Axon elongation in stiff Col-I 3D matrices was reduced (31%) following 24 h in culture compared to soft matrices. In stiff matrices, neurites fasciculated and formed less dense halos. Consistently, almost no F-actin rich growth cones were recognized, and F-actin staining was strongly reduced in the axonal compartment. This study shows that stiffness negatively affects 3D neurite outgrowth and adds insights on the cytoskeletal responses upon mechanic interactions of axons with a 3D environment. Our data will serve to facilitate the development of model systems that are mechanically well-behaved but still mimic key physiologic properties observed in vivo.Fil: Martínez, Gaby F. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Fagetti, Jimena. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Vierci, Gabriela. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Brauer, M. Mónica. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Unsain, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Richeri, Analía. Instituto de Investigaciones Biológicas "Clemente Estable"; Urugua