Through-skull fluorescence imaging of the brain in a new near-infrared window

To date, brain imaging has largely relied on X-ray computed tomography and magnetic resonance angiography with limited spatial resolution and long scanning times. Fluorescence-based brain imaging in the visible and traditional near-infrared regions (400–900 nm) is an alternative but currently requires craniotomy, cranial windows and skull thinning techniques, and the penetration depth is limited to 1–2 mm due to light scattering. Here, we report through-scalp and through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy utilizing the intrinsic photoluminescence of single-walled carbon nanotubes in the 1.3–1.4 micrometre near-infrared window. Reduced photon scattering in this spectral region allows fluorescence imaging reaching a depth of >2 mm in mouse brain with sub-10 micrometre resolution. An imaging rate of ~5.3 frames/s allows for dynamic recording of blood perfusion in the cerebral vessels with sufficient temporal resolution, providing real-time assessment of blood flow anomaly in a mouse middle cerebral artery occlusion stroke model

Longevity biotechnology:bridging AI, biomarkers, geroscience and clinical applications for healthy longevity

The recent unprecedented progress in ageing research and drug discovery brings together fundamental research and clinical applications to advance the goal of promoting healthy longevity in the human population. We, from the gathering at the Aging Research and Drug Discovery Meeting in 2023, summarised the latest developments in healthspan biotechnology, with a particular emphasis on artificial intelligence (AI), biomarkers and clocks, geroscience, and clinical trials and interventions for healthy longevity. Moreover, we provide an overview of academic research and the biotech industry focused on targeting ageing as the root of age-related diseases to combat multimorbidity and extend healthspan. We propose that the integration of generative AI, cutting-edge biological technology, and longevity medicine is essential for extending the productive and healthy human lifespan

Microarray analysis of the in vivo response of microglia to Aβ peptides in mice with conditional deletion of the prostaglandin EP2 receptor

Amyloid-β (Aβ) peptides accumulate in the brains of patients with Alzheimer's disease (AD), where they generate a persistent inflammatory response from microglia, the innate immune cells of the brain. The immune modulatory cyclooxygenase/prostaglandin E2 (COX/PGE2) pathway has been implicated in preclinical AD development, both in human epidemiology studies and in transgenic rodent models of AD [2,3]. PGE2 signals through four G-protein-coupled receptors, including the EP2 receptor that has been investigated for its role in mediating the inflammatory and phagocytic responses to Aβ [4]. To identify transcriptional differences in microglia lacking the EP2 receptor, we examined mice with EP2 conditionally deleted in Cd11b-expressing immune cells. We injected Aβ peptides or saline vehicle into the brains of adult mice, isolated primary microglia, and analyzed RNA expression by microarray. The resulting datasets were analyzed in two studies [5,6], one describing the basal status of microglia with or without EP2 deletion, and the second study analyzing the microglial response to Aβ. Here we describe in detail the experimental design and data analyses. The raw data from these studies are deposited in GEO, accession GSE57181 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57181)

Myeloid Cell Prostaglandin E2 Receptor EP4 Modulates Cytokine Production but Not Atherogenesis in a Mouse Model of Type 1 Diabetes.

Type 1 diabetes mellitus (T1DM) is associated with cardiovascular complications induced by atherosclerosis. Prostaglandin E2 (PGE2) is often raised in states of inflammation, including diabetes, and regulates inflammatory processes. In myeloid cells, a key cell type in atherosclerosis, PGE2 acts predominately through its Prostaglandin E Receptor 4 (EP4; Ptger4) to modulate inflammation. The effect of PGE2-mediated EP4 signaling specifically in myeloid cells on atherosclerosis in the presence and absence of diabetes is unknown. Because diabetes promotes atherosclerosis through increased arterial myeloid cell accumulation, we generated a myeloid cell-targeted EP4-deficient mouse model (EP4M-/-) of T1DM-accelerated atherogenesis to investigate the relationship between myeloid cell EP4, inflammatory phenotypes of myeloid cells, and atherogenesis. Diabetic mice exhibited elevated plasma PGE metabolite levels and elevated Ptger4 mRNA in macrophages, as compared with non-diabetic littermates. PGE2 increased Il6, Il1b, Il23 and Ccr7 mRNA while reducing Tnfa mRNA through EP4 in isolated myeloid cells. Consistently, the stimulatory effect of diabetes on peritoneal macrophage Il6 was mediated by PGE2-EP4, while PGE2-EP4 suppressed the effect of diabetes on Tnfa in these cells. In addition, diabetes exerted effects independent of myeloid cell EP4, including a reduction in macrophage Ccr7 levels and increased early atherogenesis characterized by relative lesional macrophage accumulation. These studies suggest that this mouse model of T1DM is associated with increased myeloid cell PGE2-EP4 signaling, which is required for the stimulatory effect of diabetes on IL-6, markedly blunts the effect of diabetes on TNF-α and does not modulate diabetes-accelerated atherogenesis

Myeloid cell EP4-deficiency does not impact atherosclerotic lesions in the aortic sinus.

<p>Representative aortic sinus lesion cross-sections stained with a Movat’s pentachrome stain from the four groups of mice are shown (A-D). Adjacent sections were immunostained for macrophages (using an anti-Mac-2 antibody; E) and smooth muscle cells (using a smooth muscle α-actin antibody; F). Cross-sectional lesion area (G), Mac-2-positive lesion area (H) and smooth muscle-positive lesion area (I) was quantified. Statistical analysis was performed by two-way ANOVA. (J-N) Twenty-four cross-sections/mouse were scored for presence or absence of left coronary sinus lesional necrotic cores and cholesterol clefts (J-K). Frequency of macrophage-rich lesions were scored for the three sinus lesions; left coronary (LC), right coronary (RC) and non-coronary (NC) (L-N). The results are presented and mean ± SEM. Data were analyzed by Kruskal-Wallis test (n = 8–15). There were no significant differences between the four groups. ND, non-diabetic; D, diabetic.</p

Myeloid cell EP4-deficiency does not alter diabetes induction, plasma lipid levels or WBC counts.

<p>The study plan in shown in A. Blood glucose levels were measured at week 0 (prior to injection of LCMV), 4, 8 and 12 by a stick test (B). Plasma cholesterol (C) and triglycerides (D) were measured by kits from Wako. Blood leukocyte counts were determined by a Hemavet (E-G). Leukocyte <i>Ptger4</i> mRNA levels were measured by real-time PCR (H). The results are presented and mean ± SEM. Data were analyzed by one-way ANOVA with Tukey's multiple comparisons test (n = 5–11 in B-C; n = 9–14 in D; 4–7 in E-G and 14–21 in H). * p<0.05; ** p<0.01; *** p<0.001; ND, non-diabetic; D, diabetic; LCMV, lymphocytic choriomeningitis virus; LFD, low-fat diet.</p

Rationale for generation of a myeloid cell-targeted EP4-deficient mouse model.

<p>Plasma and resident peritoneal macrophages were isolated from <i>Ldlr</i>^<i>-/-</i><i>; Gp</i>^<i>Tg</i> mice 12 weeks after induction of diabetes and from non-diabetic littermate controls. Glucose levels were measured in blood from the saphenous vein by a stick test (A). Plasma PGE metabolites were measured by ELISA (B). The pups from the <i>Ptger4</i>^<i>fl/fl</i> <i>x Lyz2-Cre</i>^<i>Tg/Tg</i> cross were genotyped as described in Materials and Methods (C). Resident peritoneal macrophages (rpMac) (D) and hearts (E) were harvested from EP4^M-/- mice and WT littermate controls, and <i>Ptger1-4</i> mRNA levels were measured by real-time PCR. The results are presented and mean ± SEM. Data in A-B (n = 9–22) were analyzed by unpaired two-tailed Student’s <i>t-</i>test and data in D-E were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (n = 5–7). Statistical outliers were identified by Grubbs’ test and were excluded from the analysis (one outlier in B), * p<0.05; *** p<0.001; NS, non-significant; ND, non-diabetic; D, diabetic.</p

PGE₂ exerts divergent effects on inflammatory mediators through EP4 in myeloid cells.

<p>Bone marrow-derived dendritic cells (BMDCs) and resident peritoneal macrophages from EP4^M-/- mice and WT littermates were stimulated with 10 nmol/l PGE₂ or vehicle for 8 h. <i>Il6</i> mRNA (A-B), <i>Tnfa</i> mRNA (C-D), <i>Il1b</i> mRNA (E-F), <i>Ccr7</i> mRNA (G), and <i>Il23</i> mRNA (H) were measured by real-time PCR. IL-6 (I) and TNF-α (J) release was quantified by ELISA. The results are presented and mean ± SEM. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test (real-time PCR data; 4–11; ELISA data; n = 4–6). Statistical outliers were identified by Grubbs’ test and were excluded from analyses (one outlier in A, two outliers in E), * p<0.05; ** p<0.01; *** p<0.001.</p