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

A FABP4-PPARγ signaling axis regulates human monocyte responses to electrophilic fatty acid nitroalkenes

Nitro-fatty acids (NO2-FA) are electrophilic lipid mediators derived from unsaturated fatty acid nitration. These species are produced endogenously by metabolic and inflammatory reactions and mediate anti-oxidative and anti-inflammatory responses. NO2-FA have been postulated as partial agonists of the Peroxisome Proliferator- Activated Receptor gamma (PPARγ), which is predominantly expressed in adipocytes and myeloid cells. Herein, we explored molecular and cellular events associated with PPARγ activation by NO2-FA in monocytes and macrophages. NO2-FA induced the expression of two PPARγ reporter genes, Fatty Acid Binding Protein 4 (FABP4) and the scavenger receptor CD36, at early stages of monocyte differentiation into macrophages. These responses were inhibited by the specific PPARγ inhibitor GW9662. Attenuated NO2-FA effects on PPARγ sig- naling were observed once cells were differentiated into macrophages, with a significant but lower FABP4 up- regulation, and no induction of CD36. Using in vitro and in silico approaches, we demonstrated that NO2-FA bind to FABP4. Furthermore, the inhibition of monocyte FA binding by FABP4 diminished NO2-FA-induced upre- gulation of reporter genes that are transcriptionally regulated by PPARγ, Keap1/Nrf2 and HSF1, indicating that FABP4 inhibition mitigates NO2-FA signaling actions. Overall, our results affirm that NO2-FA activate PPARγ in monocytes and upregulate FABP4 expression, thus promoting a positive amplification loop for the downstream signaling actions of this mediator

Semaforinas y neuropilinas :aportes a la plasticidad de la inervación uterina

Tribunal : Dr. Omar Trujillo-Cenoz, Dra. Patricia Cassina, Dr. Flavio Zoless

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

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

Informe final del proyecto: Riesgos en la primera infancia del consumo de cocaína en el embarazo: estudio de la inervación del cordón umbilical del recién nacido

Un equipo multidisciplinario de investigadores abordó el estudio del impacto del consumo de drogas de abuso como un factor crítico en torno a las alteraciones del desarrollo en la primera infancia Los primeros resultados obtenidos en el Instituto de Investigaciones Biológicas Clemente Estable (IIBCE) mostraron que existe una sub-población de niños recién nacidos en el Hospital de Clínicas, de madres consumidoras de cocaína que presentan una inervación simpática exacerbada en los vasos sanguíneos de su cordón umbilical. El proyecto “Riesgos en la primera infancia del consumo de cocaína en el embarazo: estudio de la inervación del cordón umbilical del recién nacido” propone correlacionar los datos generados en IIBCE con datos cuantitativos recabados por la Encuesta de Nutrición, Desarrollo Infantil y Salud (ENDIS) 2018. El establecimiento de una asociación entre variables clínico-básicas del recién nacido con los datos de la ENDIS contribuirá a la generación de políticas públicas basadas en evidencias tendientes a resolver problemas de la primera infancia en Uruguay El proyecto fue apoyado a través del Fondo Sectorial de Salud en Primera Infancia junto al Programa “Uruguay Crece Contigo” del Ministerio de Desarrollo Social.Agencia Nacional de Investigación e Innovació

Informe final del proyecto: Riesgos en la primera infancia del consumo de cocaína en el embarazo: estudio de la inervación del cordón umbilical del recién nacido

Un equipo multidisciplinario de investigadores abordó el estudio del impacto del consumo de drogas de abuso como un factor crítico en torno a las alteraciones del desarrollo en la primera infancia Los primeros resultados obtenidos en el Instituto de Investigaciones Biológicas Clemente Estable (IIBCE) mostraron que existe una sub-población de niños recién nacidos en el Hospital de Clínicas, de madres consumidoras de cocaína que presentan una inervación simpática exacerbada en los vasos sanguíneos de su cordón umbilical. El proyecto “Riesgos en la primera infancia del consumo de cocaína en el embarazo: estudio de la inervación del cordón umbilical del recién nacido” propone correlacionar los datos generados en IIBCE con datos cuantitativos recabados por la Encuesta de Nutrición, Desarrollo Infantil y Salud (ENDIS) 2018. El establecimiento de una asociación entre variables clínico-básicas del recién nacido con los datos de la ENDIS contribuirá a la generación de políticas públicas basadas en evidencias tendientes a resolver problemas de la primera infancia en Uruguay El proyecto fue apoyado a través del Fondo Sectorial de Salud en Primera Infancia junto al Programa “Uruguay Crece Contigo” del Ministerio de Desarrollo Social.Agencia Nacional de Investigación e Innovació