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.

Introduction: 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. Materials and Methods: 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, 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). Results: 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 because a neurotoxic effect per se was observed at higher concentrations assayed. Medium-chain triglyceride oil used in EPI formulation could explain this difference. Conclusion: Our data support a neuroprotective effect of EPI that 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 that could be critical to avoid neurotoxicity at very high doses.

Morphological evidence of the protective effects of a synthetic chalcone against the striatal myelin damage induced by glutaric acid.

Ultrastructural features of striatal white matter and cells in an in vivo model of glutaric acidemia type I created by intracerebral injection of glutaric acid (GA) were analyzed by transmission electron microscopy and immunohistochemistry. To test if the white matter damage observed in this model could be prevented, we administered the synthetic chemopreventive molecule CH38 ((E)-3-(4-methylthiophenyl)-1-phenyl-2-propen-1-one) to newborn rats, previous to an intracerebroventricular injection of GA. The study was done when striatal myelination was incipient and when it was already established (at 12 and 45 days post-injection [DPI], respectively). Results obtained indicate that that the ultrastructure of astrocytes and neurons did not appear significantly affected by the GA bolus. Instead, in oligodendrocytes, the most prominent GA-dependent injury defects included endoplasmic reticulum (ER) stress and nuclear envelope swelling at 12 DPI. Altered and reduced immunoreactivities against heavy neurofilament (NF), proteolipid protein (PLP), and myelin-associated glycoprotein (MAG) together with axonal bundle fragmentation and decreased myelin were also found at both ages analyzed. CH38 by itself did not affect striatal cells or axonal packages. However, the group of rats that received CH38 before GA did not show evidence neither of ER stress nor nuclear envelope dilation in oligodendrocytes, and axonal bundles appeared less fragmented. In this group, labeling of NF and PLP was similar to the controls. These results suggest that the CH38 molecule is a candidate drug to prevent or decrease the neural damage elicited by a pathological increase of GA in the brain. Optimization of the treatments and identification of the mechanisms underlying CH38 protective effects will open new therapeutic windows to protect myelin, which is a vulnerable target of numerous nervous system diseases.

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.

Correction to: Neuropilin-1 receptor in the rapid and selective estrogen-induced neurovascular remodeling of rat uterus.

The article title of the original publication contains error for the term "estrogen" was captured twice.

Neuropilin-1 receptor in the rapid and selective estrogen-induced neurovascular remodeling of rat uterus.

Sympathetic nerves innervate most organs and regulate organ blood flow. Specifically, in the uterus, estradiol (E2) elicits rapid degeneration of sympathetic axons and stimulates the growth of blood vessels. Both physiological remodeling processes, critical for reproduction, have been extensively studied but as independent events and are still not fully understood. Here, we examine the neuropilin-1 (NRP1), a shared receptor for axon guidance and angiogenic factors. Systemic estradiol or vehicle were chronically injected to prepubertal rats and uterine and sympathetic chain sections immunostained for NRP1. Uterine semaphorin-3A mRNA was evaluated by in situ hybridization. Control sympathetic uterine-projecting neurons (1-month-old) expressed NRP1 in their somas but not in their intrauterine terminal axons. Estradiol did not affect NRP1 in the distal ganglia. However, at the entrance of the organ, some sympathetic NRP1-positive nerves were recognized. Vascular NRP1 was confined to intrauterine small-diameter vessels in both hormonal conditions. Although the overall pattern of NRP1-IR was not affected by E2 treatment, a subpopulation of infiltrated eosinophil leukocytes showed immunoreactivity for NRP1. Sema3A transcripts were detected in this cellular type as well. No NRP1-immunoreactive axons nor infiltrated eosinophils were visualized in other estrogenized pelvic organs. Together, these data suggest the involvement of NRP1/Sema3A signaling in the selective E2-induced uterine neurovascular remodeling. Our data support a model whereby NRP1 could coordinate E2-induced uterine neurovascular remodeling, acting as a positive regulator of growth when expressed in vessels and as a negative regulator of growth when expressed on axons.

Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy.

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodic skeleton (MPS) is conserved across animals, suggesting it is a fundamental component of neuronal extensions. The nanoscale architecture of the arrangement (190 nm) is below the resolution limit of conventional fluorescent microscopy. Fluorescent nanoscopy, on the other hand, requires costly equipment and special analysis routines, which remain inaccessible to most research groups. This report aims to resolve this issue by using protein-retention expansion microscopy (pro-ExM) to reveal the MPS of axons. ExM uses reagents and equipment that are readily accessible in most neurobiology laboratories. We first explore means to accurately estimate the expansion factors of protein structures within cells. We then describe the protocol that produces an expanded specimen that can be examined with any fluorescent microscopy allowing quantitative nanoscale characterization of the MPS. We validate ExM results by direct comparison to stimulated emission depletion (STED) nanoscopy. We conclude that ExM facilitates three-dimensional, multicolor and quantitative characterization of the MPS using accessible reagents and conventional fluorescent microscopes.

Author Correction: Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.

Reinnervation of rat endometrium in the anterior eye chamber model of experimental endometriosis: Old methods for new questions.

Endometriosis is a benign estrogen-dependent chronic gynecological disease characterized by the presence of endometrial-like tissue outside the uterine cavity. In both women and experimental endometriotic rats, endometriosis lesions endow autonomic and sensory nerves, which are thought to contribute to the disease-associated pain. Some evidence indicates that the reinnervation of lesions is regulated by factors produced by the endometrial tissue as well as by environmental factors from the peritoneum. In this study, we examined the reinnervation of the rat endometrial tissue in an ectopic environment different from the peritoneum employing the anterior eye chamber model of experimental endometriosis. At 3 and 6weeks following transplantation, endometrial grafts retained many histological features of the eutopic tissue. Both sympathetic and sensory nerves reinnervated endometrial grafts and distributed in the stroma-like tissue, around blood vessels and in close proximity to the glands and lining epithelium. Sympathetic innervation was more robust than sensory innervation. No significant topographical relationship between sympathetic nerves and macrophages was observed. These results suggest that the rat endometrium possesses intrinsic neuritogenic capacities and can be reinnervated by sympathetic and sensory nerves in ectopic sites different from the peritoneum.

Estrogen up-regulation of semaphorin 3F correlates with sympathetic denervation of the rat uterus.

Current evidence indicates that rises in systemic levels of estrogen create in the uterus an inhibitory environment for sympathetic nerves. However, molecular insights of these changes are far from complete. We evaluated if semaphorin 3F mRNA, a sympathetic nerve repellent, was produced by the rat uterus and if its expression was modulated by estrogen. We also analyzed whether uterine nerves express the semaphorin 3F binding receptor, neuropilin-2. Uterine levels of semaphorin 3F mRNA were measured using real time reverse transcriptase-polymerase chain reaction in prepubertal rat controls and following chronic estrogen treatment. Localization of semaphorin 3F transcripts was determined by in situ hybridization and the expression of neuropilin-2 was assessed by immunohistochemistry. These studies showed that: (1) chronic estrogen treatment led to a 5-fold induction of semaphorin 3F mRNA in the immature uterus; (2) estrogen provoked a tissue-specific induction of semaphorin 3F which was particularly localized in the connective tissue that borders muscle bundles and surrounds intrauterine blood vessels; (3) two major cell-types were recognized in the areas where transcripts were concentrated, fibroblast-like cells and infiltrating eosinophil leukocytes; and (4) some delicate nerve terminal profiles present in the estrogenized uterus were immunoreactive for neuropilin-2. Temporal and spatial expression patterns of semaphorin 3F/neuropilin-2 are consistent with a possible role of this guidance cue in the remodeling of uterine sympathetic innervation by estrogen. Though correlative in its nature, these data support a model whereby semaphorin 3F, in combination with other inhibitory molecules, converts the estrogenized myometrium to an inhospitable environment for sympathetic nerves.