Δ9-tetrahydrocannabinol and 2-AG decreases neurite outgrowth and differentially affects ERK1/2 and Akt signaling in hiPSC-derived cortical neurons.

Endocannabinoids regulate different aspects of neurodevelopment. In utero exposure to the exogenous psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC), has been linked with abnormal cortical development in animal models. However, much less is known about the actions of endocannabinoids in human neurons. Here we investigated the effect of the endocannabinoid 2-arachidonoyl glycerol (2AG) and Δ9-THC on the development of neuronal morphology and activation of signaling kinases, in cortical neurons derived from human induced pluripotent stem cells (hiPSCs). Our data indicate that the cannabinoid type 1 receptor (CB1R), but not the cannabinoid 2 receptor (CB2R), GPR55 or TRPV1 receptors, is expressed in young, immature hiPSC-derived cortical neurons. Consistent with previous reports, 2AG and Δ9-THC negatively regulated neurite outgrowth. Interestingly, acute exposure to both 2AG and Δ9-THC inhibited phosphorylation of serine/threonine kinase extracellular signal-regulated protein kinases (ERK1/2), whereas Δ9-THC also reduced phosphorylation of Akt (aka PKB). Moreover, the CB1R inverse agonist SR 141716A attenuated the decrease in neurite outgrowth and ERK1/2 phosphorylation induced by 2AG and Δ9-THC. Taken together, our data suggest that hiPSC-derived cortical neurons express CB1Rs and are responsive to exogenous cannabinoids. Thus, hiPSC-neurons may represent a good cellular model for investigating the role of the endocannabinoid system in regulating cellular processes in developing human neurons.

Single-molecule fluorescence in-situ hybridization reveals that human SHANK3 mRNA expression varies during development and in autism-associated SHANK3 heterozygosity.

BACKGROUND: Deletions and mutations in the SHANK3 gene are strongly associated with autism spectrum disorder and underlie the autism-associated disorder Phelan-McDermid syndrome. SHANK3 is a scaffolding protein found at the post-synaptic membrane of excitatory neurons. METHODS: Single-molecule fluorescence in-situ hybridization (smFISH) allows the visualization of single mRNA transcripts in vitro. Here we perform and quantify smFISH in human inducible pluripotent stem cell (hiPSC)-derived cortical neurons, targeting the SHANK3 transcript. RESULTS: Both smFISH and conventional immunofluorescence staining demonstrated a developmental increase in SHANK3 mRNA and protein, respectively, in control human cortical neurons. Analysis of single SHANK3 mRNA molecules in neurons derived from an autistic individual heterozygous for SHANK3 indicated that while the number of SHANK3 mRNA transcripts remained comparable with control levels in the cell soma, there was a 50% reduction within neuronal processes, suggesting that local, dendritic targeting of SHANK3 mRNA may be specifically affected in SHANK3 haploinsufficiency. CONCLUSION: Human SHANK3 mRNA shows developmentally regulated dendritic localization in hiPSC-derived neurons, which is reduced in neurons generated from a haploinsufficient individual with autism. Although further replication is needed, given the importance of local mRNA translation in synaptic function, this could represent an important early abnormality.

Rapid Modulation of Axon Initial Segment Length Influences Repetitive Spike Firing.

Neurons implement a variety of plasticity mechanisms to alter their function over timescales ranging from seconds to days. One powerful means of controlling excitability is to directly modulate the site of spike initiation, the axon initial segment (AIS). However, all plastic structural AIS changes reported thus far have been slow, involving days of neuronal activity perturbation. Here, we show that AIS plasticity can be induced much more rapidly. Just 3 hr of elevated activity significantly shortened the AIS of dentate granule cells in a calcineurin-dependent manner. The functional effects of rapid AIS shortening were offset by dephosphorylation of voltage-gated sodium channels, another calcineurin-dependent mechanism. However, pharmacological separation of these phenomena revealed a significant relationship between AIS length and repetitive firing. The AIS can therefore undergo a rapid form of structural change over timescales that enable interactions with other forms of activity-dependent plasticity in the dynamic control of neuronal excitability.

Rod microglia: a morphological definition.

Brain microglial morphology relates to function, with ramified microglia surveying the micro-environment and amoeboid microglia engulfing debris. One subgroup of microglia, rod microglia, have been observed in a number of pathological conditions, however neither a function nor specific morphology has been defined. Historically, rod microglia have been described intermittently as cells with a sausage-shaped soma and long, thin processes, which align adjacent to neurons. More recently, our group has described rod microglia aligning end-to-end with one another to form trains adjacent to neuronal processes. Confusion in the literature regarding rod microglia arises from some reports referring to the sausage-shaped cell body, while ignoring the spatial distribution of processes. Here, we systematically define the morphological characteristics of rod microglia that form after diffuse brain injury in the rat, which differ morphologically from the spurious rod microglia found in uninjured sham. Rod microglia in the diffuse-injured rat brain show a ratio of 1.79 ± 0.03 cell length:cell width at day 1 post-injury, which increases to 3.35 ± 0.05 at day 7, compared to sham (1.17 ± 0.02). The soma length:width differs only at day 7 post-injury (2.92 ± 0.07 length:width), compared to sham (2.49 ± 0.05). Further analysis indicated that rod microglia may not elongate in cell length but rather narrow in cell width, and retract planar (side) processes. These morphological characteristics serve as a tool for distinguishing rod microglia from other morphologies. The function of rod microglia remains enigmatic; based on morphology we propose origins and functions for rod microglia after acute neurological insult, which may provide biomarkers or therapeutic targets.

Objective Morphological Quantification of Microscopic Images Using a Fast Fourier Transform (FFT) Analysis.

Quantification of immunohistochemistry (IHC) and immunofluorescence (IF) using image intensity depends on a number of variables. These variables add a subjective complexity in keeping a standard within and between laboratories. Fast Fourier Transformation (FFT) algorithms, however, allow for a rapid and objective quantification (via statistical analysis) using cell morphologies when the microscopic structures are oriented or aligned. Quantification of alignment is given in terms of a ratio of FFT intensity to the intensity of an orthogonal angle, giving a numerical value of the alignment of the microscopic structures. This allows for a more objective analysis than alternative approaches, which rely upon relative intensities.

Rod microglia: elongation, alignment, and coupling to form trains across the somatosensory cortex after experimental diffuse brain injury.

BACKGROUND: Since their discovery, the morphology of microglia has been interpreted to mirror their function, with ramified microglia constantly surveying the micro-environment and rapidly activating when changes occur. In 1899, Franz Nissl discovered what we now recognize as a distinct microglial activation state, microglial rod cells (Stäbchenzellen), which he observed adjacent to neurons. These rod-shaped microglia are typically found in human autopsy cases of paralysis of the insane, a disease of the pre-penicillin era, and best known today from HIV-1-infected brains. Microglial rod cells have been implicated in cortical 'synaptic stripping' but their exact role has remained unclear. This is due at least in part to a scarcity of experimental models. Now we have noted these rod microglia after experimental diffuse brain injury in brain regions that have an associated sensory sensitivity. Here, we describe the time course, location, and surrounding architecture associated with rod microglia following experimental diffuse traumatic brain injury (TBI). METHODS: Rats were subjected to a moderate midline fluid percussion injury (mFPI), which resulted in transient suppression of their righting reflex (6 to 10 min). Multiple immunohistochemistry protocols targeting microglia with Iba1 and other known microglia markers were undertaken to identify the morphological activation of microglia. Additionally, labeling with Iba1 and cell markers for neurons and astrocytes identified the architecture that surrounds these rod cells. RESULTS: We identified an abundance of Iba1-positive microglia with rod morphology in the primary sensory barrel fields (S1BF). Although present for at least 4 weeks post mFPI, they developed over the first week, peaking at 7 days post-injury. In the absence of contusion, Iba1-positive microglia appear to elongate with their processes extending from the apical and basal ends. These cells then abut one another and lay adjacent to cytoarchitecture of dendrites and axons, with no alignment with astrocytes and oligodendrocytes. Iba1-positive rod microglial cells differentially express other known markers for reactive microglia including OX-6 and CD68. CONCLUSION: Diffuse traumatic brain injury induces a distinct rod microglia morphology, unique phenotype, and novel association between cells; these observations entice further investigation for impact on neurological outcome.

Inhibition of oral carcinogenesis by citrus flavonoids.

Six citrus flavonoids were tested for antineoplastic activity. The hamster cheek pouch model was utilized, and the solutions of the flavonoids (2.0-2.5%) and the solution of the carcinogen, 7,12-dimethylbenz[a]anthracene (0.5%), were applied topically to the pouches. The pouches of the positive controls were treated with the solvent used to dissolve the flavonoids and the solution of the carcinogen. The data show that 4 flavonoids (hesperetin, neohesperidin, tangeretin, and nobiletin) were inactive. The results with naringin and naringenin show that both of these flavonoids significantly lowered tumor number [5.00 (control group), 2.53 (naringin group), and 3.25 (naringenin group)]. Naringin also significantly reduced tumor burden [269 mm(3)(control group) and 77.1 mm(3)(naringin group)]. The data suggest that naringin and naringenin, 2 flavonoids found in high concentrations in grapefruit, may be able to inhibit the development of cancer.

Evaluation of preemptive valdecoxib therapy on initial archwire placement discomfort in adults.

The purpose of this randomized, double-blinded, placebo-controlled prospective clinical trial was to compare the efficacy of preemptive and postoperative administration of valdecoxib in reducing discomfort caused by initial archwire placement in adults. A total of 56 orthodontic patients aged 18-54 years who were to begin treatment were randomly assigned to one of three groups: (1) placebo, (2) those who received preemptive valdecoxib 40 mg at least 30 minutes before initial archwire placement, or (3) those who received postoperative valdecoxib 40 mg two hours after initial archwire placement. Patients in the active treatment groups also received continuous valdecoxib therapy for an additional 48 hours. Discomfort levels were recorded on a visual analog scale at zero, two, six, 24, and 48 hours after initial archwire placement. At baseline, no significant differences were detected between the three groups. In the preemptive valdecoxib group, there was no significant increase (P > .05) in discomfort from baseline at any time point. The placebo and postoperative valdecoxib groups showed significant (P < .05) increases in discomfort after six hours, with the peak discomfort at 24 hours. The postoperative group showed a tendency toward decreased discomfort over time, but the changes were not significantly different from the placebo or the preemptive group. Preemptive analgesia with nonsteroidal anti-inflammatory drugs may be an approach to prevent discomfort associated with initial archwire placement in healthy adults.

Efficacy and economic evaluation of pilocarpine in treating radiation-induced xerostomia.

Xerostomia, resulting from therapeutic irradiation of the head and neck, can be debilitating and destructively impact the patient's quality of life. The first priority of treatment of radiation-induced xerostomia should be to preserve oral and dental health. Therapeutic doses of fluoride and meticulous dental hygiene are the treatments of choice for this purpose. Measures taken to prevent superinfection with cariogenic bacteria and fungi are also important. In addition to minimising damage to the dentition, it is important to provide palliative therapy to help the patient live a more normal life. Salivary substitutes help some patients but muscarinic, cholinergic sialogogues provide a more physiological replacement in those patients who have measurable residual salivary function. An oral formulation of pilocarpine hydrochloride is approved to treat radiation-induced xerostomia. In such cases, it has been shown to effectively stimulate salivary flow and relieve subjective symptoms associated with oral dryness. Administration of this drug is associated with predictable, but generally tolerable, adverse effects. Similar drugs, approved for other indications, have been shown to produce comparable results. These drugs are generally less expensive than the oral formulation of pilocarpine. As treatment of irradiation-induced xerostomia with muscarinic, cholinergic sialogogues is palliative, not curative, administration of these drugs should be individualised and guided by the patient's willingness to balance symptomatic efficacy with adverse effects and the expense of treatment.

The prevalence of periodontal-related changes in adolescents with asthma: results of the Third Annual National Health and Nutrition Examination Survey.

PURPOSE: The purpose of this study was to explore the association between asthma and periodontal disease in adolescents using oral examination and health interview data from the Third National Health and Nutrition Examination Survey (NHANES III) 1988-1994. METHODS: The study population comprised 1,596 adolescents 13 to 17 years of age: 253 (16%) asthmatics and 1,358 (84%) nonasthmatic controls who were examined for bleeding on probing (BOP), subgingival calculus (SBC), supragingival calculus (SPC), probing depth greater than or equal to 3 mm (PD), and loss of periodontal attachment greater than or equal to 2 mm (LPA). The authors fitted separate multivariate GEE Poisson regression models adjusting for parents' income, gender, race, exposure to potentially xerogenic drugs (antihistamines, corticosteroids, and inhalers), tobacco exposure, and dental examination within the past year. RESULTS: None of the periodontal measures was associated with asthma severity or with the use of antiasthmatic drugs. However, several covariates had statistically significant odds ratios (P < .05). CONCLUSIONS: There was no evidence to support the association between asthma and periodontal health in the adolescent population. Since the findings may be due to the inherent limitations of cross-sectional studies, the lack of knowledge about the daily dose of antiasthmatic medication, and the level of-compliance with the therapeutic regimen, future studies should be longitudinal and monitor medication use.