Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization

© 2019 The Author(s). Background: Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases. Methods: In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples. Results: Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model. Conclusions: Advantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided

Improved long-term survival with subdural drains following evacuation of chronic subdural haematoma

Background Chronic subdural haematoma (CSDH) is a common condition that is effectively managed by burrhole drainage but requires repeat surgery in a significant minority of patients. The Cambridge Chronic Subdural Haematoma Trial (CCSHT) was a randomised controlled study that showed placement of subdural drains for 48 h following burrhole evacuation significantly reduces the incidence of reoperation and improves survival at 6 months. The present study examined the long-term survival of the patients in the trial. Methods In the original trial patients at a single neurosurgical centre from 2004–2007 were randomly assigned to receive a drain (n = 108) or no drain (n = 107) following burrhole drainage of CSDH. We ascertained whether the trial patients were alive in February 2016—a minimum of 8 years following enrollment—via the UK NHS tracing service. Survival was compared between the trial groups and against expected survival for the UK general population matched for age and sex. Results At 5 years following surgery the drain group continued to have significantly better survival than the no drain patients (p = 0.027), but this was no longer apparent at 10 years. Survival of patients in the drain group did not differ significantly from that of the general population whereas patients who did not receive a drain had significantly lower survival than expected (p = 0.0006). Conclusion Subdural drains following CSDH evacuation are associated with improved long-term survival, which appears similar to that expected for the general population of the same age and sex. All patients having burrhole CSDH evacuation should receive a drain as standard practice unless specifically contraindicated

TLR 2 and 4 responsiveness from isolated peripheral blood mononuclear cells from rats and humans as potential chronic pain biomarkers

Background: Chronic pain patients have increased peripheral blood mononuclear cell Interkeukin-1β production following TLR2 and TLR4 simulation. Here we have used a human-to-rat and rat-to-human approach to further investigate whether peripheral blood immune responses to TLR agonists might be suitable for development as possible systems biomarkers of chronic pain in humans. Methods and Results: Study 1: using a graded model of chronic constriction injury in rats, behavioral allodynia was assessed followed by in vitro quantification of TLR2 and TLR4 agonist-induced stimulation of IL-1β release by PBMCs and spinal cord tissues (n = 42; 6 rats per group). Statistical models were subsequently developed using the IL-1β responses, which distinguished the pain/no pain states and predicted the degree of allodynia. Study 2: the rat-derived statistical models were tested to assess their predictive utility in determining the pain status of a published human cohort that consists of a heterogeneous clinical pain population (n = 19) and a pain-free population (n = 11). The predictive ability of one of the rat models was able to distinguish pain patients from controls with a ROC AUC of 0.94. The rat model was used to predict the presence of pain in a new chronic pain cohort and was able to accurately predict the presence of pain in 28 out of the 34 chronic pain participants. Conclusions: These clinical findings confirm our previous discoveries of the involvement of the peripheral immune system in chronic pain. Given that these findings are reflected in the prospective graded rat data, it suggests that the TLR response from peripheral blood and spinal cord were related to pain and these clinical findings do indeed act as system biomarkers for the chronic pain state. Hence, they provide additional impetus to the neuroimmune interaction to be a drug target for chronic pain.Yuen H. Kwok, Jonathan Tuke, Lauren L. Nicotra, Peter M. Grace, Paul E. Rolan, Mark R. Hutchinso

A record-linkage study of the development of hepatocellular carcinoma in persons with hepatitis C infection in Scotland

We investigated trends in first time hospital admissions and deaths attributable to hepatocellular carcinoma (HCC) in a large population based cohort of 22 073 individuals diagnosed with hepatitis C viral (HCV) infection through laboratory testing in Scotland in 1991 2006. We identified new cases of HCC through record linkage to the national inpatient hospital discharge database and deaths registry. A total of 172 persons diagnosed with HCV were admitted to hospital or died with first time mention of HCC. Hepatocellular carcinoma incidence increased between 1996 and 2006 (average annual change of 6.1, 95% confidence interval (CI):0.9 11.6%, P¼0.021). The adjusted relative risk of HCC was greater for males (hazard ratio¼2.7, 95% CI: 1.7 4.2), for those aged 60 years or older (hazard ratio ¼2.7, 95% CI: 1.9 4.1) compared with 50 59 years, and for those with a previous alcohol related hospital admission (hazard ratio¼2.5, 95% CI: 1.7 3.7). The risk of individuals diagnosed with HCV developing HCC was greatlyincreased compared with the general Scottish population (standardised incidence ratio¼127, 95% CI: 102 156). Owing to the advancing age of the Scottish HCV diagnosed population, the annual number of HCC cases is projected to increase, with a consequent increasing burden on the public healthcare system

Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91phox subunit of NADPH oxidase

<p>Abstract</p> <p>Background</p> <p>Both (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner. Multiple lines of evidence have documented a pivotal role of overactivated NADPH oxidase (NOX2) in inflammation-mediated neurodegeneration. We hypothesized that NOX2 might be a novel action site of naloxone to mediate its anti-inflammatory actions.</p> <p>Methods</p> <p>Inhibition of NOX-2-derived superoxide by (-) and (+)-naloxone was measured in lipopolysaccharide (LPS)-treated midbrain neuron-glia cultures and phorbol myristate acetate (PMA)-stimulated neutrophil membranes by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt (WST-1) or ferricytochrome c. Further, various ligand (³H-naloxone) binding assays were performed in wild type and gp91<it>^phox-/-</it>neutrophils and transfected COS-7 and HEK293 cells. The translocation of cytosolic subunit p47<it>^phox</it>to plasma membrane was assessed by western blot.</p> <p>Results</p> <p>Both (-) and (+)-naloxone equally inhibited LPS- and PMA-induced superoxide production with an IC50 of 1.96 and 2.52 μM, respectively. Competitive binding of ³H-naloxone with cold (-) and (+)-naloxone in microglia showed equal potency with an IC50 of 2.73 and 1.57 μM, respectively. ³H-Naloxone binding was elevated in COS-7 and HEK293 cells transfected with gp91^{<it>phox</it>}; in contrast, reduced ³H-naloxone binding was found in neutrophils deficient in gp91^{<it>phox </it>}or in the presence of a NOX2 inhibitor. The specificity and an increase in binding capacity of ³H-naloxone were further demonstrated by 1) an immunoprecipitation study using gp91^{<it>phox </it>}antibody, and 2) activation of NOX2 by PMA. Finally, western blot studies showed that naloxone suppressed translocation of the cytosolic subunit p47^{<it>phox </it>}to the membrane, leading to NOX2 inactivation.</p> <p>Conclusions</p> <p>Strong evidence is provided indicating that NOX2 is a non-opioid novel binding site for naloxone, which is critical in mediating its inhibitory effect on microglia overactivation and superoxide production.</p

The influence of the frequency of periodic disturbances on the maintenance of phytoplankton diversity

The influence of periodic disturbances of various frequency on the maintenance of the phytoplankton diversity was studied by semicontinuous competition experiments. Disturbances consisted of dilution events, which meant both addition of fresh nutrients and elimination of organisms. The intervals between dilution events varied from 1 to 14 days. Diversity was found to increase with increasing intervals between disturbances. coexisting species belonged to different strategy types: (a) species with rapid growth under enriched conditions, (b) species with good competitive abilities under impoverished conditions, (c) species with the ability to build up storage pools of the limiting nutrient. An increase of the number of coexisting species over the number that would have coexisted in steady state was only found when the interval exceeded one generation time

Succinate supplementation improves metabolic performance of mixed glial cell cultures with mitochondrial dysfunction

Mitochondrial dysfunction, the inability to efficiently utilise metabolic fuels and oxygen, contributes to pathological changes following traumatic spinal cord or traumatic brain injury (TBI). In the present study, we tested the hypothesis that succinate supplementation can improve cellular energy state under metabolically stressed conditions in a robust, reductionist in vitro model of mitochondrial dysfunction in which primary mixed glial cultures (astrocytes, microglia and oligodendrocytes) were exposed to the mitochondrial complex I inhibitor rotenone. Cellular response was determined by measuring intracellular ATP, extracellular metabolites (glucose, lactate, pyruvate), and oxygen consumption rate (OCR). Rotenone produced no significant changes in glial ATP levels. However, it induced metabolic deficits as evidenced by lactate/pyruvate ratio (LPR) elevation (a clinically-established biomarker for poor outcome in TBI) and decrease in OCR. Succinate addition partially ameliorated these metabolic deficits. We conclude that succinate can improve glial oxidative metabolism, consistent our previous findings in TBI patients' brains. The mixed glial cellular model may be useful in developing therapeutic strategies for conditions involving mitochondrial dysfunction, such as TBI

Three-Dimensional Imaging-Based Web Application for Predicting Tracheal Tube Depth in Preterm Neonates

BACKGROUND: Positioning a tracheal tube (TT) to the correct depth in preterm infants is challenging. Currently, there is no reliable single-predictor model for neonates applicable to the whole range of size or age. OBJECTIVE: In this study, we used post-mortem magnetic resonance imaging (PMMRI) of preterm infants to measure tracheal dimensions and to develop a clinical guide for TT positioning. METHODS: We measured tracheal length (TL) and tracheal diameter (TD) in a cohort of normal neonates and foetuses that underwent PMMRI (cause of death unexplained). The distance between the lips and the mid-tracheal point, i.e., the mid-tracheal length (mid-TL), and the TD measurement were obtained. We produced univariate prediction models of mid-TL and TD, using gestational age (GA), foot length (FL), crown-rump length (CRL) and body weight (BW) as potential predictors, as well as multiple prediction models for mid-TL. RESULTS: Tracheal measurements were performed in 117 cases, with a mean GA of 28.8 weeks (range 14-42 weeks). The best linear association was between mid-TL and FL (mid-TL = FL × 0.914 + 1.859; R2 = 0.94), but was improved by multivariate regression models. We developed a prediction tool using only GA and BW (R2 = 0.92), and all four predictors (GA, BW, FL and CRL; R2 = 0.94) which is now available as a web-based application via the Internet. CONCLUSION: Post-mortem imaging data provide estimates of TT insertion depth. Our prediction tool based on age and BW can be used at the bedside and is ready to be tested in clinical practice

Shikonin Increases Glucose Uptake in Skeletal Muscle Cells and Improves Plasma Glucose Levels in Diabetic Goto-Kakizaki Rats

Glucose is the most common substrate for energy metabolism. Despite the varying demands for glucose, the body needs to regulate its internal environment and maintain a constant and stable condition. Glucose homeostasis requires harmonized interaction between several tissues, achieving equilibrium between glucose output and uptake. In this thesis we aimed to investigate factors modulating glucose homeostasis in a rat model of type 2 diabetes, the Goto-Kakizaki (GK) rat. In addition, we investigated sex differences in hepatic carbohydrate and lipid metabolism in healthy rats. In Paper I, three-week but not three-day treatment with a Southeast Asian herb, Gynostemma pentaphyllum (GP), significantly reduced plasma glucose (PG) levels in GK rats. An intra-peritoneal glucose tolerance test (IPGTT) was significantly improved in GP-treated compared to placebo-treated group. In the GP treated rats, the glucose response in an intra-peritoneal pyruvate tolerance test was significantly lower, indicating decreased gluconeogenesis, and hepatic glucose output (HGO) was reduced. GP-treatment significantly reduced hepatic glycogen content, but not glycogen synthase activity. The study provides evidence that the GP extract exerted anti-diabetic effect in GK rats, reducing PG levels and HGO, suggesting that GP improves the hepatic insulin sensitivity by suppressing gluconeogenesis. In Paper II, shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increased glucose uptake in L6 myotubes, but did not phosphorylate Akt. Furthermore we found no evidence for the involvement of AMP activated protein kinase (AMPK) in shikonin induced glucose uptake. Shikonin increased the intracellular levels of calcium in these cells and stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myotubes. In GK rats treated with shikonin once daily for 4 days, PG levels were significantly decreased. In an insulin sensitivity test, the absolute PG levels were significantly lower in the shikonin-treated rats. These findings suggest that shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium. In Paper III, GK and control Wistar rats were injected daily for up to 4 weeks with either a non-hematopoietic erythropoietin analog ARA290 or with placebo. PG levels in GK but not Wistar rats were significantly lower in ARA290-treated compared to placebo. After 2 and 4 weeks, the IPGTT was significantly improved in ARA290 treated GK rats. In insulin and pyruvate tolerance tests, glucose responses were similar in ARA290 and placebo groups. In isolated GK rat islets, glucose-stimulated insulin release was two-fold higher and islet intracellular calcium concentrations in response to several secretagogues were significantly higher in ARA290-treated than in placebo-treated GK rats. These findings indicate that treatment with ARA290 significantly improved glucose tolerance in diabetic GK rats, most likely due to improvement of insulin release. In Paper IV, sex differences in hepatic carbohydrate and lipid metabolism were characterized in healthy rats. No sex-differences were observed regarding hepatic triglyceride content, fatty acid oxidation rates or insulin sensitivity. Male rats had higher ratios of insulin to glucagon levels, increased hepatic glycogen content, a lower degree of AMPK phosphorylation, a higher rate of glucose production and higher expression levels of gluconeogenic genes, as compared to female rats. A sex-dependent response to mild starvation was observed with males being more sensitive. In conclusion, sex-differences reflect a higher capacity of the healthy male rat liver to respond to increased energy demands. Key words: glucose homeostasis, type 2 diabetes, GK rats, L6 myotubes, hepatic glucose output, insulin sensitivity, sex differences

Visibility Representations of Boxes in 2.5 Dimensions

We initiate the study of 2.5D box visibility representations (2.5D-BR) where vertices are mapped to 3D boxes having the bottom face in the plane $z=0$ and edges are unobstructed lines of sight parallel to the $x$- or $y$-axis. We prove that: $(i)$ Every complete bipartite graph admits a 2.5D-BR; $(ii)$ The complete graph $K_n$ admits a 2.5D-BR if and only if $n \leq 19$; $(iii)$ Every graph with pathwidth at most $7$ admits a 2.5D-BR, which can be computed in linear time. We then turn our attention to 2.5D grid box representations (2.5D-GBR) which are 2.5D-BRs such that the bottom face of every box is a unit square at integer coordinates. We show that an $n$-vertex graph that admits a 2.5D-GBR has at most $4n - 6 \sqrt{n}$ edges and this bound is tight. Finally, we prove that deciding whether a given graph $G$ admits a 2.5D-GBR with a given footprint is NP-complete. The footprint of a 2.5D-BR $\Gamma$ is the set of bottom faces of the boxes in $\Gamma$.Comment: Appears in the Proceedings of the 24th International Symposium on Graph Drawing and Network Visualization (GD 2016