Early postnatal exposure to isoflurane causes cognitive deficits and disrupts development of newborn hippocampal neurons via activation of the mTOR pathway

Funding: Johns Hopkins ACCM Department anesthesiology.hopkinsmedicine.org (grant number StAAR) to CDM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NIH-NIGMS www.nih.gov (grant number 1R01GM120519-01 and 1K08GM104329-01) to CDM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NIH www.nih.gov (grant number NS048271 and MH105128) to GLM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NIH www.nih.gov (grant number NS047344) to HS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments: We would like to acknowledge the helpful contributions of Sunu Kim (technical assistance) and Allan Gottschalk (critical commentary).Peer reviewedPublisher PD

Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model

Abstract In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib’s effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1–10 µM and 25 mg/kg) and in combination with doxil (10–100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell–cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a − 53% versus − 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib’s ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities

Higher mitochondrial DNA copy number is associated with lower prevalence of microalbuminuria

It has been suggested that mitochondrial dysfunction contributes to the initiation and development of atherosclerosis and cardiovascular disease. We examined the association between mitochondrial DNA (mtDNA) copy number and microalbuminuria in a cross-sectional community-based study. We measured peripheral blood mtDNA copy number in 694 adults without chronic kidney disease by a real-time PCR method. The overall prevalence of microalbuminuria (defined as an albumin creatinine ratio of 30 to 299 mg/g) was 4.5%. The prevalence of microalbuminuria decreased progressively from the lower to the upper quartiles of mtDNA copy number (6.9%, 5.7%, 2.9%, and 2.3% in quartiles 1, 2, 3, and 4, respectively, P = 0.017 for trend). Multiple logistic regression models showed that the quartile of mtDNA copy number was independently associated with the prevalence of microalbuminuria (P = 0.01 for trend). Compared with the lowest quartile, the highest quartile had an odds ratio of 0.22 for microalbuminuria (95% confidence interval, 0.05 to 0.87; P = 0.03). Higher mtDNA copy number was associated with the lower prevalence of microalbuminuria in a community-based population

Isoflurane exposure leads to aberrant activation of the mechanistic target of rapamycin (mTOR) signaling pathway, and pharmacological inhibition of the mTOR activities rescues deficits in behavioral tests and loss of spines.

<p>(A) Representative confocal images of phospho-S6 (pS6) immunofluorescence at postnatal day (P) 30 in the dentate gyrus showing an increase in labeling in the isoflurane plus vehicle (Iso/V) group relative to controls and a return to baseline in the group exposed to isoflurane and subsequently treated with rapamycin, designated Iso/R. The upper panels are original confocal images with DAPI in blue and pS6 labeling in red, and the lower panels are processed for quantification with black pS6 signal on white background (ML, molecular layer; DG, dentate gyrus; HI, hilus, scale bar: 50 μm). Also shown in (A) quantification of normalized pS6 expression in the dentate gyrus granule cell layer (***<i>p</i> < 0.001, ANOVA, numbers in each bar represent n for images analyzed). (B) Schematic diagram of rapamycin treatment for behavior tests and spine analysis. Summaries of total dendritic length (C) and Sholl analysis of dendritic complexity (D) of GFP+ neurons show a rescue of normal dendritic arbor length and complexity with Iso/R. Values represent mean ± SEM (*<i>p</i> < 0.05, **<i>p</i> < 0.01; ANOVA for C; *<i>p</i> < 0.0001 ANOVA for D). Numbers in each bar represent number of cells analyzed per group, minimum of 5 animals per group). Summaries of object-place recognition test (E) and Y-maze test (F) for Iso/V and Iso/R show a recovery to near control performance with Iso/R. (Control <i>n</i> = 10, Iso/V <i>n</i> = 11, Iso/R <i>n</i> = 11; *: <i>p</i> < 0.05; **: <i>p</i> < 0.01, Student <i>t</i> test). (G) Representative confocal images of dendritic spines at P60. Scale bar: 2 μm. Shown on right are summary plots of total and mature dendritic spine density. Numbers associated with bar graph indicate the number of dendritic segments examined, a total of 2,586 spines in the control group, 1,831 spines in the isoflurane plus vehicle group, and 2,999 spines in the isoflurane plus rapamycin group were analyzed (****<i>p</i> < 0.0001; ns: non-significant; ANOVA, numbers in each bar represent n of dendritic segments analyzed per group, minimum of 5 animals per group). Underlying data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001246#pbio.2001246.s008" target="_blank">S1 Data</a> under Fig 3A-G.</p

Isoflurane exposure results in overgrowth of dendritic arbors.

<p>(A) A schematic diagram of isoflurane exposure procedure for morphology examination. (B) Sample confocal image of dentate gyrus granule cell (DGCs) infected with retrovirus expressing green florescent protein (GFP) (scale bar: 100 μm). Representative confocal images (C) and tracings (D) of individual control and isoflurane-exposed GFP+ neurons at postnatal day (P) 30 exhibiting overgrowth in the isoflurane group relative to control conditions (scale bar: 10 μm for both C and D). Summaries of total dendritic length (E) and Sholl analysis of dendritic complexity (F) of GFP+ neurons show marked overgrowth of dendritic arbors. Numbers associated with bar graph indicate the number of neurons examined from at least 5 animals per group. The same groups of neurons were examined in (E) and (F). Values represent mean ± SEM (**<i>p</i> < 0.01; Student <i>t</i> test for E and *<i>p</i> < 0.0001 ANOVA for F). Underlying data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001246#pbio.2001246.s008" target="_blank">S1 Data</a> under Fig 1F tab.</p