Mapping acute neuroinflammation in vivo with diffusion-MRI in rats given a systemic lipopolysaccharide challenge.

It is becoming increasingly apparent that neuroinflammation plays a critical role in an array of neurological and psychiatric disorders. Recent studies have demonstrated the potential of diffusion MRI (dMRI) to characterize changes in microglial density and morphology associated with neuroinflammation, but these were conducted mostly ex vivo and/or in extreme, non-physiological animal models. Here, we build upon these studies by investigating the utility of well-established dMRI methods to detect neuroinflammation in vivo in a more clinically relevant animal model of sickness behavior. We show that diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) indicate widespread increases in diffusivity in the brains of rats given a systemic lipopolysaccharide challenge (n = 20) vs. vehicle-treated controls (n = 12). These diffusivity changes correlated with histologically measured changes in microglial morphology, confirming the sensitivity of dMRI to neuroinflammatory processes. This study marks a further step towards establishing a noninvasive indicator of neuroinflammation, which would greatly facilitate early diagnosis and treatment monitoring in various neurological and psychiatric diseases.

Resolving the cellular specificity of TSPO imaging in a rat model of peripherally-induced neuroinflammation.

The increased expression of 18 kDa Translocator protein (TSPO) is one of the few available biomarkers of neuroinflammation that can be assessed in humans in vivo by positron emission tomography (PET). TSPO PET imaging of the central nervous system (CNS) has been widely undertaken, but to date no clear consensus has been reached about its utility in brain disorders. One reason for this could be because the interpretation of TSPO PET signal remains challenging, given the cellular heterogeneity and ubiquity of TSPO in the brain. The aim of the current study was to ascertain if TSPO PET imaging can be used to detect neuroinflammation induced by a peripheral treatment with a low dose of the endotoxin, lipopolysaccharide (LPS), in a rat model (ip LPS), and investigate the origin of TSPO signal changes in terms of their cellular sources and regional distribution. An initial pilot study utilising both [18F]DPA-714 and [11C]PK11195 TSPO radiotracers demonstrated [18F]DPA-714 to exhibit a significantly higher lesion-related signal in the intracerebral LPS rat model (ic LPS) than [11C]PK11195. Subsequently, [18F]DPA-714 was selected for use in the ip LPS study. Twenty-four hours after ip LPS, there was an increased uptake of [18F]DPA-714 across the whole brain. Further analyses of regions of interest, using immunohistochemistry and RNAscope Multiplex fluorescence V2 in situ hybridization technology, showed TSPO expression in microglia, monocyte derived-macrophages, astrocytes, neurons and endothelial cells. The expression of TSPO was significantly increased after ip LPS in a region-dependent manner: with increased microglia, monocyte-derived macrophages and astrocytes in the substantia nigra, in contrast to the hippocampus where TSPO was mostly confined to microglia and astrocytes. In summary, our data demonstrate the robust detection of peripherally-induced neuroinflammation in the CNS utilising the TSPO PET radiotracer, [18F]DPA-714, and importantly, confirm that the resultant increase in TSPO signal increase arises mostly from a combination of microglia, astrocytes and monocyte-derived macrophages.

Emergent Medical Illnesses Related to Cannabis Use.

As more states rush to legalize the use of cannabis products, both medically and recreationally, there are more medical harms being seen in emergency departments (ED). The tetrahydrocannabinol (THC) concentration today is much stronger than the concentration from the 90s. In the 1990s most typical "joints" contained 1-3 mg of THC. Today, plants are being raised and modified to produce a higher concentration of THC. In turn, the amount of cannabidiol (CBD) is decreasing. Previously, people would smoke 1-3 mg of THC. The typical joint in Colorado contains 18 mg of THC or more. Currently, in the ED, we see patients who self-report smoking 2,000 mg or more of THC in a day. In 2015, 2.6 million individuals started cannabis use, 45% were 12-17 years of age.3 This brief report includes some of the more common illnesses that have been seen over the last four years of legalization in Colorado, and is by no means inclusive of all the potential problems that can occur. Among the many untoward effects being seen, illnesses that will be discussed are: cannabinoid associated hyperemesis, acute psychosis, cannabinoid catatonia syndrome, acute myo-pericarditis and ingestions.

The effects of acute Methylene Blue administration on cerebral blood flow and metabolism in humans and rats.

Methylene Blue (MB) is a brain-penetrating drug with putative neuroprotective, antioxidant and metabolic enhancing effects. In vitro studies suggest that MB enhances mitochondrial complexes activity. However, no study has directly assessed the metabolic effects of MB in the human brain. We used in vivo neuroimaging to measure the effect of MB on cerebral blood flow (CBF) and brain metabolism in humans and in rats. Two doses of MB (0.5 and 1 mg/kg in humans; 2 and 4 mg/kg in rats; iv) induced reductions in global cerebral blood flow (CBF) in humans (F(1.74, 12.17)5.82, p = 0.02) and rats (F(1,5)26.04, p = 0.0038). Human cerebral metabolic rate of oxygen (CMRO2) was also significantly reduced (F(1.26, 8.84)8.01, p = 0.016), as was the rat cerebral metabolic rate of glucose (CMRglu) (t = 2.6(16) p = 0.018). This was contrary to our hypothesis that MB will increase CBF and energy metrics. Nevertheless, our results were reproducible across species and dose dependent. One possible explanation is that the concentrations used, although clinically relevant, reflect MB's hormetic effects, i.e., higher concentrations produce inhibitory rather than augmentation effects on metabolism. Additionally, here we used healthy volunteers and healthy rats with normal cerebral metabolism where MB's ability to enhance cerebral metabolism might be limited.

Erbb4 Deletion From Inhibitory Interneurons Causes Psychosis-Relevant Neuroimaging Phenotypes.

BACKGROUND AND HYPOTHESIS: Converging lines of evidence suggest that dysfunction of cortical GABAergic inhibitory interneurons is a core feature of psychosis. This dysfunction is thought to underlie neuroimaging abnormalities commonly found in patients with psychosis, particularly in the hippocampus. These include increases in resting cerebral blood flow (CBF) and glutamatergic metabolite levels, and decreases in ligand binding to GABAA α5 receptors and to the synaptic density marker synaptic vesicle glycoprotein 2A (SV2A). However, direct links between inhibitory interneuron dysfunction and these neuroimaging readouts are yet to be established. Conditional deletion of a schizophrenia susceptibility gene, the tyrosine kinase receptor Erbb4, from cortical and hippocampal inhibitory interneurons leads to synaptic defects, and behavioral and cognitive phenotypes relevant to psychosis in mice. STUDY DESIGN: Here, we investigated how this inhibitory interneuron disruption affects hippocampal in vivo neuroimaging readouts. Adult Erbb4 conditional mutant mice (Lhx6-Cre;Erbb4F/F, n = 12) and their wild-type littermates (Erbb4F/F, n = 12) were scanned in a 9.4T magnetic resonance scanner to quantify CBF and glutamatergic metabolite levels (glutamine, glutamate, GABA). Subsequently, we assessed GABAA receptors and SV2A density using quantitative autoradiography. RESULTS: Erbb4 mutant mice showed significantly elevated ventral hippccampus CBF and glutamine levels, and decreased SV2A density across hippocampus sub-regions compared to wild-type littermates. No significant GABAA receptor density differences were identified. CONCLUSIONS: These findings demonstrate that specific disruption of cortical inhibitory interneurons in mice recapitulate some of the key neuroimaging findings in patients with psychosis, and link inhibitory interneuron deficits to non-invasive measures of brain function and neurochemistry that can be used across species.

Relevance of sleep and associated structural changes in GBA1 mouse to human rapid eye movement behavior disorder.

Rapid eye movement (REM) sleep behaviour disorder (RBD) is a REM parasomnia that often predicts the later occurrence of alpha-synucleinopathies. Variants in the gene encoding for the lysosomal enzyme glucocerebrosidase, GBA, strongly increase the risk of RBD. In a GBA1-mouse model recently shown to mimic prodromal stages of α-synucleinopathy, we now demonstrate striking REM and NREM electroencephalographic sleep abnormalities accompanied by distinct structural changes in the more widespread sleep neurocircuitry.

In vivo multi-parametric manganese-enhanced MRI for detecting amyloid plaques in rodent models of Alzheimer's disease.

Amyloid plaques are a hallmark of Alzheimer's disease (AD) that develop in its earliest stages. Thus, non-invasive detection of these plaques would be invaluable for diagnosis and the development and monitoring of treatments, but this remains a challenge due to their small size. Here, we investigated the utility of manganese-enhanced MRI (MEMRI) for visualizing plaques in transgenic rodent models of AD across two species: 5xFAD mice and TgF344-AD rats. Animals were given subcutaneous injections of MnCl2 and imaged in vivo using a 9.4 T Bruker scanner. MnCl2 improved signal-to-noise ratio but was not necessary to detect plaques in high-resolution images. Plaques were visible in all transgenic animals and no wild-types, and quantitative susceptibility mapping showed that they were more paramagnetic than the surrounding tissue. This, combined with beta-amyloid and iron staining, indicate that plaque MR visibility in both animal models was driven by plaque size and iron load. Longitudinal relaxation rate mapping revealed increased manganese uptake in brain regions of high plaque burden in transgenic animals compared to their wild-type littermates. This was limited to the rhinencephalon in the TgF344-AD rats, while it was most significantly increased in the cortex of the 5xFAD mice. Alizarin Red staining suggests that manganese bound to plaques in 5xFAD mice but not in TgF344-AD rats. Multi-parametric MEMRI is a simple, viable method for detecting amyloid plaques in rodent models of AD. Manganese-induced signal enhancement can enable higher-resolution imaging, which is key to visualizing these small amyloid deposits. We also present the first in vivo evidence of manganese as a potential targeted contrast agent for imaging plaques in the 5xFAD model of AD.

Morphine-induced nitric oxide production in isolated, iris-ciliary bodies.

Considerable evidence suggests that the nitric oxide (NO)/cGMP signaling pathway plays an integral role in opioid receptor-mediated responses in the cardiovascular and immune systems. Previous studies in our laboratory and others have shown that nitric oxide (NO) plays a role in morphine-induced reduction of intraocular pressure (IOP) and pupil diameter (PD) in the New Zealand white (NZW) rabbit. The present study is designed to determine the effect of morphine on NO production in the isolated, iris-ciliary body (ICB), site of aqueous humor production, as this effect could be associated with morphine-stimulated changes in aqueous humor dynamics and iris function. ICBs obtained from normal NZW rabbits were utilized in these experiments. In some experiments, ICB samples were treated with morphine (1, 10 and 100 microM) for 1 h and later examined for changes in NO levels using a NO detection kit. In other experiments, tissue samples were pretreated with naloxone (non-selective opioid receptor antagonist), L-NAME (non-selective NO-synthase inhibitor) or GSH (sulfhydryl reagent) for 30 min, followed by treatment with morphine (10 muM). Morphine caused a concentration-dependent increase in the release of NO from ICBs. Levels of NO detected in the incubation medium of ICB samples increased from 1.49 +/- 0.19 (control) to 8.81 +/- 2.20 microM/mg protein (morphine-treated; 100 microM). Morphine-stimulated release of NO was significantly inhibited in tissues pretreated with 10 microM naloxone, L-NAME, or GSH. Results obtained from this study suggest that morphine stimulates NO release from the ICB through a mechanism that involves activation of NO-releasing opioid receptors. These results support the in vivo effects of morphine demonstrated in previous studies.

Effects of Resveratrol on Inflammatory Biomarkers in Glaucomatous Human Trabecular Meshwork Cells.

PURPOSE: Resveratrol (RSV), an antioxidant polyphenol, has demonstrated beneficial effects in various ocular diseases including glaucoma. Our study was designed to evaluate the effects of RSV on nitric oxide synthase (NOS) enzymes, nitric oxide (NO) and interleukin-1 alpha (IL-1 α), in human glaucomatous trabecular meshwork (TM) cells. METHODS: Western blot was utilized to determine endothelial and inducible NOS (eNOS, iNOS) expression. The concentration-related effects of RSV on IL-1 α and NO levels were assessed using the respective ELISA kits. RESULTS: Densitometry data showed concentration-related increases in eNOS, and reduction in iNOS expression at high RSV concentrations. RSV treatment (0.1, 1, 10 and 100 µM) resulted in increased NO levels (6 ± 0.7, 7 ± 0.8, 7.3 ± 0.7 and 9.5 ± 1 nM/mg protein, respectively). The average value obtained for control was 4.8 ± 0.6 nM/mg protein. Significant increases in IL-1α levels were observed with lower concentrations of RSV. However, at higher RSV concentrations (10-100 µM), IL-1 levels decreased. CONCLUSIONS: Resveratrol increased NO in glaucomatous TM cells, possibly by increasing eNOS expression. Thus, RSV-induced NO production supports the beneficial effects of this antioxidant in glaucoma. Furthermore, our results showing a reduction in iNOS, a contributor to oxidative stress expression, further support RSV's antioxidant capabilities in vision.

Quercetin reduces hydroxyurea induced cytotoxicity in immortalized mouse aortic endothelial cells.

BACKGROUND: Chronic inflammation is a characteristic of sickle cell disease (SCD), and is invariably associated with vascular endothelial injury. Hydroxyurea (HU), a naturally cytotoxic chemotherapeutic agent, is the only FDA drug approved for SCD, and is therefore naturally cytotoxic. Quercetin (QCT) is a dietary flavonoid found ubiquitously in plants and foods that have anti-oxidative and anti-inflammatory characteristics. Our hypothesis is that dietary QCT will decrease cytotoxic effects of lipopolysaccharide (LPS) and HU induced vascular cell damage. METHODS: Lipopolysaccharide (LPS) was used to induce inflammation in immortalized mouse aortic endothelial cells (iMAECs), providing an in vitro model of inflamed endothelial cells. The cells were exposed to LPS throughout the entire experiment. Interventions included treating the LPS exposed cells with QCT, HU, or QCT + HU over 50 hours. The 50-hour period included 24 hours of varying treatments, followed by two hours of hypoxic exposure and then 24 hours under normal aerobic exposure. RESULTS: LDH level was significantly higher for LPS treated versus untreated cells (P = 0.0004). LPS plus 30 micromole QCT reduced the LDH (p = 0.1, trend), whereas LPS plus 100 micromoles HU, significantly increased LDH (p = 0.0004). However, LPS plus treatment with 30 micromoles QCT/100 micromoles HU, significantly reduced LDH, compared with HU alone (p = 0.0002). DISCUSSION: These results suggest that quercetin may be effective against vascular endothelial cell damage for iMAECs in vitro. In particular, it shows promise in preventing HU-induced cytotoxicity, surprisingly found from these results. This latter finding is important, and should be given more consideration, since HU is the only FDA-approved drug for treating sickle cell patients, and its use is rapidly increasing.

Screening urine samples for the absence of urinary tract infection using the sediMAX automated microscopy analyser.

Urinalysis culminates in a workload skew within the clinical microbiology laboratory. Routine processing involves screening via manual microscopy or biochemical dipstick measurement, followed by culture for each sample. Despite this, as many as 80% of specimens are reported as negative; thus, there is vast wastage of resources and time, as well as delayed turnaround time of results as numerous negative cultures fulfil their required incubation time. Automation provides the potential for streamlining sample screening by efficiently (>30% sample exclusion) and reliably [negative predictive value (NPV) ≥ 95%] ruling out those likely to be negative, whilst also reducing resource usage and hands-on time. The present study explored this idea by using the sediMAX automated microscopy urinalysis platform. We prospectively collected and processed 1411 non-selected samples directly after routine laboratory processing. The results from this study showed multiple optimum cut-off values for microscopy. However, although optimum cut-off values permitted rule-out of 40.1% of specimens, an associated 87.5% NPV was lower than the acceptable limit of 95%. Sensitivity and specificity of leukocytes and bacteria in determining urinary tract infection was assessed by receiver operator characteristic curves with area under the curve values found to be 0.697 [95% confidence interval (CI): 0.665-0.729] and 0.587 (95% CI: 0.551-0.623), respectively. We suggested that the sediMAX was not suitable for use as a rule-out screen prior to culture and further validation work must be carried out before routine use of the analyser.

Kappa opioid receptor localization and coupling to nitric oxide production in cells of the anterior chamber.

PURPOSE: The present study was designed to determine whether kappa opioid receptors (KORs) are localized to cells of the inflow and outflow pathways of the eye and if activation of these receptors has an effect on nitric oxide (NO) production, because these effects could play a role in KOR agonist-mediated reduction of IOP. METHODS: Human nonpigmented ciliary epithelial (NPCE) and trabecular meshwork (HTM-3) cells were treated with spiradoline (SPR), a selective KOR agonist, or estradiol, for 24 hours. Some cells were pretreated with the selective KOR antagonist norbinaltorphimine (norBNI) or the nonselective NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) for 30 minutes, followed by the addition of SPR. Immunofluorescent localization of KORs was determined in isolated rabbit iris-ciliary bodies (ICBs) and NPCE and HTM-3 cells. RESULTS: Immunohistochemical data show the localization of KORs to the rabbit ICB and more specifically to the ciliary epithelial layer. KORs were also found on cell membranes of NPCE and HTM-3 cells. Treatment of both these cell types with spiradoline caused concentration-dependent increases in the release of NO. Spiradoline-induced release of NO from both cell types was inhibited by pretreatment with norBNI and L-NAME. CONCLUSIONS: Results from this study show the presence of KORs on rabbit ICBs and also on NPCE and HTM cells. Activation of these KORs on both cell types resulted in KOR-mediated increases in NO production. These findings provide evidence that previously demonstrated KOR-mediated reduction in IOP could be caused, in part, by NO production in both the ciliary body and the trabecular meshwork.