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

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 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 inhibits LPS-induced cytokines 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 2 h, and then for an additional 6 h in the presence or absence of 5 ng/ml LPS. <i>Il6</i> mRNA (A-B), <i>Tnfa</i> mRNA (C-D), and <i>Il1b</i> mRNA (E) were measured by real-time PCR. TNF-α (F-G) and IL-6 (H-I) release was quantified by ELISA. The results are presented as fold over WT cells incubated in the absence of LPS as mean ± SEM. Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (n = 9–11). * p<0.05; ** p<0.01; *** p<0.001.</p