General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex

Calcium signaling represents the principle pathway by which astrocytes respond to neuronal activity. General anesthetics are routinely used in clinical practice to induce a sleep-like state, allowing otherwise painful procedures to be performed. Anesthetic drugs are thought to mainly target neurons in the brain and act by suppressing synaptic activity. However, the direct effect of general anesthesia on astrocyte signaling in awake animals has not previously been addressed. This is a critical issue, because calcium signaling may represent an essential mechanism through which astrocytes can modulate synaptic activity. In our study, we performed calcium imaging in awake head-restrained mice and found that three commonly used anesthetic combinations (ketamine/xylazine, isoflurane, and urethane) markedly suppressed calcium transients in neocortical astrocytes. Additionally, all three anesthetics masked potentially important features of the astrocyte calcium signals, such as synchronized widespread transients that appeared to be associated with arousal in awake animals. Notably, anesthesia affected calcium transients in both processes and soma and depressed spontaneous signals, as well as calcium responses, evoked by whisker stimulation or agonist application. We show that these calcium transients are inositol 1,4,5-triphosphate type 2 receptor (IP₃R2)-dependent but resistant to a local blockade of glutamatergic or purinergic signaling. Finally, we found that doses of anesthesia insufficient to affect neuronal responses to whisker stimulation selectively suppressed astrocyte calcium signals. Taken together, these data suggest that general anesthesia may suppress astrocyte calcium signals independently of neuronal activity. We propose that these glial effects may constitute a nonneuronal mechanism for sedative action of anesthetic drugs.Astrocytes display both spontaneous and evoked increases in cytosolic calcium (Ca²⁺) in response to a variety of stimuli (1). These calcium transients are thought to represent a fundamental type of astrocyte signaling involved in modulating a range of vital brain functions, including cerebral blood flow, synaptic activity, cell volume, and extracellular ion homeostasis (2–10). General anesthesia is known to both depress and alter normal neuronal firing patterns in the cortex (11, 12). Conversely, the effects of anesthesia on astrocyte function have not been systematically explored.The molecular mechanisms by which anesthetic drugs suppress consciousness are incompletely understood. Almost every general anesthetic has numerous molecular targets in the brain, yet they all produce a remarkably similar suppression and synchronization of neuronal activity (13). In our study, we chose to test three widely used anesthetics thought to act on quite different neuronal targets: (i) isoflurane, (ii) a combination of ketamine and xylazine, and (iii) urethane. Isoflurane is believed to potentiate γ-aminobutyric acid type A receptor (GABA_AR) chloride currents and decrease glutamatergic activity both in the cortex and subcortical centers (14). Ketamine principally suppresses N-methyl-d-aspartic acid (NMDA)-mediated excitatory signaling in the cortex and subcortical centers. Ketamine is frequently used in combination with the α₂-adrenoreceptor agonist xylazine to enhance sedative effects by suppressing sympathetic activity (15). Finally, urethane (ethyl carbamate) has diffuse effects on both inhibitory and excitatory neurotransmission, as well as a range of ion channels (16). However, it is currently not known whether nonneuronal targets, such as astrocytes, may mediate some of the sedative effects of these anesthetics.Our study addresses whether general anesthetics also affect astrocyte calcium signaling. To explore this question, we applied general anesthesia to awake head-restrained mice while simultaneously measuring calcium signals in neocortical astrocytes by two-photon laser-scanning microscopy (2PLSM) and neuronal activity with an extracellular electrocorticogram (ECoG) electrode (11, 17). We found that all three anesthetics potently suppressed astrocyte calcium transients in both the cell soma and fine processes. Moreover, the three anesthetics also fundamentally altered the pattern of calcium signals, by selectively desynchronizing astrocyte calcium transients in different cells. Additionally, we show that low doses of anesthetics selectively suppressed astrocyte calcium response to whisker stimulation, before having any effect on the amplitude of the neuronal responses. In fact, we found that local neuronal activity may not be necessary for spontaneous astrocyte calcium signaling, as blocking neuronal responses with either sodium-channel blocker tetrodotoxin (TTX), NMDA antagonist amino-5-phosphonovaleric acid (AP5), or AMPA antagonist 6-cyano-7nitroquinoxaline-2,3-dione (CNQX) did not eliminate astrocyte signals. Finally, by microinjecting adenosine triphosphate (ATP), we showed that anesthesia also reduces astrocyte calcium responses to direct agonist application. In conclusion, our study demonstrates that astrocyte calcium signals are highly sensitive to anesthetics, suggesting that the sedative actions of anesthetics could be mediated, in part, by a suppression of astrocyte calcium signaling.     

Towards dual inhibitors of the MET kinase and WNT signaling pathway; design,synthesis and biological evaluation

Both the kinase MET and the WNT signaling pathway are attractive targets in cancer therapy, and synergistic effects have previously been observed in animal models upon simultaneous inhibition. A strategy towards a designed multiple ligand of MET and WNT signaling is pursued based on the two hetero biaryl systems present in both the MET inhibitor tepotinib and WNT signaling inhibitor TC-E 5001. Initial screening was conducted to find the most suitable ring systems for further optimization, whereas a second screen explored modifications towards pyridazinones and triazolo pyridazines. Up to 54% reduction of WNT signaling activity at 10 μM concentration was achieved, however, only low affinities towards MET were observed. Overall, the thiophene substituted pyridazinone 40 was the best dual MET and WNT signaling inhibitor, with a 17% and 19% reduction of activity, respectively. Although further optimizations are needed to achieve more potent dual inhibitors, the strategy presented herein can be valuable towards the development of a dual inhibitor of MET and WNT signaling.

Dual inhibitors of MET and WNT signaling may have synergistic effects, and the design, synthesis and evaluation of such first-in-class small-molecules are reported.     

The presence of TP53 mutation at diagnosis of follicular lymphoma identifies a high-risk group of patients with shortened time to disease progression and poorer overall survival

The International Prognostic Index and the Follicular Lymphoma International Prognostic Index are widely used for the risk assessment of follicular lymphoma (FL). Although molecular studies have provided insight into the biology of FL, no molecular marker has impacted on treatment stratification. Because TP53 mutations are associated with poor prognosis in hematologic malignancies, we investigated the prognostic value of TP53 mutation at diagnosis in FL. Heterozygous TP53 mutation was detected in 12 of 185 (6%) analyzed cases. Mutation was associated with older age (P = .02) and higher International Prognostic Index score (P = .04). On multivariate analysis, TP53 mutation correlated with shorter progression-free survival (P < .001) and overall survival (P = .009). TP53 mutation was associated with low expression of the immune-response 1 gene expression signature (P = .016) and with an unfavorable gene expression-based survival predictor score (P < .001), demonstrating for the first time that molecular features of the malignant cell may correlate with the nature of the immune response in FL.     

Proinflammatory cytokines and complement activation in salvaged blood from abdominal aortic aneurism surgery and total hip replacement surgery

BACKGROUND: Levels of proinflammatory mediators in unwashed salvaged blood from abdominal aortic aneurism (AAA) surgery are unknown. We hypothesized that there are higher levels of these mediators in unwashed blood salvaged in AAA surgery compared to hip replacement surgery. STUDY DESIGN AND METHODS: Ten patients scheduled for AAA surgery (Group A) and 10 patients for total hip replacement surgery (Group H) were included. Blood samples from the autotransfusion set were obtained during surgery and arterial samples before, during, and 6 hours after surgery. Determination of interleukin (IL)‐1β, IL‐6, IL‐8, tumor necrosis factor‐α, activated complement 3 (C3a), and high‐sensitivity C‐reactive protein (CRP) were performed. Salvaged blood was not retransfused. RESULTS: Levels (median [range]) of IL‐8 in blood in the salvage system were higher in Group A versus Group H (215.3 [22.5‐697.2] vs. 35.3 [16.7‐66.6] pg/mL; p = 0.002). Higher levels of IL‐6 were also seen in Group A versus Group H (60.0 [52.6‐62.2] vs. 42.34 [19.4‐62.2] pg/mL; p = 0.049). Levels of IL‐6 in blood sampled during surgery were approximately fivefold higher in Group A versus Group H (p = 0.023), whereas approximately 70% higher levels of C3a were observed in Group H versus Group A (p = 0.021). Postoperative concentrations of IL‐1β (p = 0.002), IL‐6 (p = 0.001), and IL‐8 (0.005) were higher in Group A versus Group H. CONCLUSION: Salvaged blood in AAA surgery contains substantially higher levels of proinflammatory mediators compared to blood in total hip replacement surgery.     

Side population cells in highly enriched CD34-positive cells from peripheral blood progenitor cells identify an immature subtype of hematopoietic progenitor cells but do not predict time to engraftment in patients treated with high-dose therapy

Objective: A Hoechst 33342 dye efflux assay can be used to define a population of immature hematopoietic progenitor cells (HPC) that are called side population (SP) cells. Previously, SP cells examined from bone marrow (BM) and peripheral blood progenitor cells (PBPC) were found to be predominantly CD34 negative. Methods and results: In this study, we show that the level of CD34+ cells within the SP fraction increases from 2% in BM to 15% in mobilized PBPC. Furthermore, SP cells are found in highly enriched CD34+ cells from both BM and PBPC, and these cells define an immature phenotype of HPC. We also observed a higher level of CD133+ cells within the SPCD34+ cell population. Moreover, the frequency of long‐term culture‐initiating cells (LTC‐IC) was markedly increased in SPCD34+ cells. To further investigate whether variations in the level of SP cells in the CD34+ cell fraction influenced short‐term engraftment, we studied 20 patients with Hodgkin lymphoma that were autotransplanted with highly enriched CD34+ cells from PB. The percentage of SP cells in the PBCD34+ cell fraction was highly variable, ranging from 0.3 to 22%. No correlation was found between the content of SP cells in the autotransplanted CD34+ cells and time to short‐term engraftment. Conclusion: SPCD34+ cells in PBPC define an immature phenotype of HPC with increased numbers of LTC‐IC, and they are more frequently found in PBPC than in BM. The number of SP cells does not predict time to engraftment.     

SRD5A2 is associated with increased cortisol metabolism in schizophrenia spectrum disorders

Objective

Dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis is documented in bipolar disorder and schizophrenia, but the mechanism is unclear; recently, increased activity of cortisol metabolizing enzymes was indicated in these disorders. We investigated whether five genes involved in cortisol metabolism were associated with altered activity of cortisol metabolizing enzymes in bipolar disorder (BD) and schizophrenia spectrum disorders (SCZ).

Methods

A case–control sample of subjects with BD (N = 213), SCZ (N = 274) and healthy controls (N = 370) from Oslo, Norway, were included and genotyped from 2003 to 2008. A sub-sample (healthy controls: N = 151; SCZ: N = 40; BD: N = 39) had estimated enzyme activities based on measurements of urinary free cortisol, urinary free cortisone and metabolites. A total of 102 single nucleotide polymorphisms (SNPs) in the SRD5A1, SRD5A2, AKR1D1, HSD11B1 and HSD11B2 genes were genotyped, and significant SNPs analyzed in the sub-sample.

Results

There was a significant association of rs6732223 in SRD5A2 (5α-reductase) with SCZ (p = 0.0043, Bonferroni corrected p = 0.030, T risk allele). There was a significantly increased 5α-reductase activity associated with rs6732223 (T allele) within the SCZ group (p = 0.011).

Conclusions

The present data suggest an interaction between SCZ and SRD5A2 variants coding for the enzyme 5α-reductase, giving rise to increased 5α-reductase activity in SCZ. The findings may have implications for cortisol metabolizing enzymes as possible drug targets.     

Effect of bariatric surgery on sulphur amino acids and glutamate

Plasma total cysteine (tCys) concentrations are associated with BMI. To study the relationship between tCys and BMI, we monitored the changes in serum concentrations of tCys and metabolically related compounds in sixty obese patients (BMI 50-60 kg/m(2)) from before to 1 year after either gastric bypass surgery (mean 30 % weight loss) or duodenal switch surgery (mean 41 % weight loss). A total of fifty-eight healthy persons (BMI 17-31 kg/m(2)) served as controls. Before surgery, obese patients had modestly (approximately 17 %) higher mean serum tCys, and markedly (>2-fold) higher glutamate concentrations, than controls (P ≤ 0·001 for both). Serial examinations after surgery revealed that gastric bypass patients had no change in tCys concentrations (P = 0·22), while duodenal switch patients showed a modest (approximately 12 %) but significant decrease in tCys (P < 0·001). Total homocysteine concentrations increased in duodenal switch patients but not in gastric bypass patients. Independent of surgery type, serum concentrations of methionine and cystathionine decreased (P < 0·05 for both), while serum glutathione and taurine remained stable. Glutamate concentrations declined, as did γ-glutamyltransferase activity (P < 0·001 for both). These results show that despite 30 % weight loss, and decreases in methionine, cystathionine and glutamate, there was no significant change in serum tCys in patients after gastric bypass surgery. The decrease in tCys in patients undergoing duodenal switch could be related to malabsorption. The present findings do not suggest that BMI is a causal determinant of plasma tCys.