BACKGROUND: This study aimed to develop a modified histochemical staining technique to successfully identify arterial and venous segments of brain microvessels.
METHODS: Gelatin/red ink-alkaline phosphatase-oil red O (GIAO) staining was developed from the traditional gelatin-ink perfusion method. Oil red Chinese ink for brush writing and painting mixed with gelatin was used to label cerebral vascular lumens. Subsequently, alkaline phosphatase staining was used to label endothelial cells on the arterial segments of cerebral microvessels. Thereafter, the red ink color in vessel lumens was highlighted with oil red O staining.
RESULTS: The arterial segments of the brain microvessels exhibited red lumens surrounded by dark blue walls, while the venous segments were bright red following GIAO staining. Meanwhile, the nerve fiber bundles were stained brownish-yellow, and the nuclei appeared light green under light microscope. After cerebral infarction, we used GIAO staining to determine angiogenesis features and detected notable vein proliferation inside the infarct core. Moreover, GIAO staining in conjunction with hematoxylin staining was performed to assess the infiltration of foamy macrophages.
CONCLUSIONS: Red Chinese ink enabled subsequent multiple color staining on brain section. Oil red O was introduced to improved the resolution and contrast between arterial and venous segments of microvessels.
CONCLUSIONS: With excellent resolution, GIAO staining effectively distinguished arterial and venous segments of microvessels in both normal and ischemic brain tissue. GIAO staining, as described in the present study, will be useful for histological investigations of microvascular bed alterations in a variety of brain disorders.

With the aging population, coronary syndrome is one of the leading causes of mortality. Atherosclerosis is the pathophysiological basis of coronary syndrome, which is caused by plaque rupture and predisposed or aggravated by many perioperative complications. Parecoxib is one of the most widely used nonsteroidal anti-inflammatory perioperative drugs. This study aims to evaluate the potential benefits of parecoxib on atherosclerosis progression. Apolipoprotein E-deficient (Apo E-/-) mice were intraperitoneally injected by parecoxib (par group) or saline (control group) and, meanwhile, were given a western diet for 12 weeks. The aorta and aortic root were examined by oil red O (ORO) staining for atherosclerotic lesions. The expression level of matrix metalloproteinases (MMPs), was investigated using immunofluorescence and western blot. Macrophage inflammation was investigated by Q-PCR. Parecoxib treatment increased the number of vascular smooth muscle cells (VSMC) and amount of collagen, while and decreased the number of macrophages in murine aortic walls. The expression of MMP1, 2, 9, and 13 as well as IL- 1β and IL-6 were also decreased in the par group. However, there was no statistical difference in lipid infiltration between the two groups. Parecoxib could improve plaque stability by suppressing inflammation and inhibiting MMPs production.

Macrophages would engulf circulating oxidized (ox)- low-density lipoprotein and form lipid droplet-laden foam cells. Macrophage foam cells are considered an important therapeutic target of atherosclerosis. The aim of the study was to investigate a hypoxic foam cell model for anti-atherosclerotic drug screening using the chemical hypoxia-mimicking agent cobalt chloride (CoCl2). The oil red O stating results showed that treatment with CoCl2 could induce lipid accumulation and lead to cell transformation to spindle-shaped and lipid-rich foam cells in RAW 264.7 macrophages. Incubation with 150 μM CoCl2 for 24 h significantly increased the area of intracellular lipid droplets in macrophages, compared with the control group. Our findings indicate that CoCl2-triggered macrophage foam cells should be a potential in vitro hypoxia model for atherosclerosis drug discovery.

Syringaresinol-4-O-β-d-glucoside (SSG), a furofuran-type lignan, was found to modulate lipid and glucose metabolism through an activity screen of lipid accumulation and glucose consumption, and was therefore considered as a promising candidate for the prevention and treatment of metabolic disorder, especially in lipid and glucose metabolic homeostasis. In this study, the effects of SSG on lipogenesis and glucose consumption in HepG2 cells and C2C12 myotubes were further investigated. Treatment with SSG significantly inhibited lipid accumulation by oil red O staining and reduced the intracellular contents of total lipid, cholesterol and triglyceride in HepG2 cells. No effect was observed on cell viability in the MTT assay at concentrations of 0.1-10 μmol/L. SSG also increased glucose consumption by HepG2 cells and glucose uptake by C2C12 myotubes. Furthermore, real-time quantitative PCR revealed that the beneficial effects were associated with the down-regulation of sterol regulatory element-binding proteins-1c, -2 (SREBP-1c, -2), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC) and hydroxyl methylglutaryl CoA reductase (HMGR), and up-regulation of peroxisome proliferator-activated receptors alpha and gamma (PPARα and PPARγ). SSG also significantly elevated transcription activity of PPARγ tested by luciferase assay. These results suggest that SSG is an effective regulator of lipogenesis and glucose consumption and might be a candidate for further research in the prevention and treatment of lipid and glucose metabolic diseases.

OBJECTIVE: We sought to devise and test a multifunctional contrast dye agent for X-ray based digital radiography (DR) or computer tomography (CT), magnetic resonance imaging (MRI), and colored staining in ex vivo validation part of animal experiments.
METHODS: The custom-formulated contrast dye namely red iodized oil (RIO) was prepared by solubilizing a lipophilic dye Oil Red O in iodized poppy seed oil (Lipiodol or LPD) followed by physicochemical characterizations. To explore and test the utility of RIO, normal rats (n = 10) and rabbits (n = 10) with myocardial infarction (MI) were euthanized by overdose of pentobarbital for infusion of RIO through catheterization. The bodies and/or excised organs including heart, liver, spleen, kidneys, pancreas, and intestines of the rats and rabbits were imaged at clinical mammography, CT and MRI units. These images were qualitatively studied and quantitatively analyzed using Wilcoxon Rank test with a P value < 0.05 being considered of a statistically significant difference. Imaging findings were verified by histomorphology.
RESULTS: All experimental procedures were carried out successfully with the use of RIO. T1 and T2 relaxation time was 234.2 ± 2.6 ms and 141.9 ± 3.0 ms for RIO, close to that of native LPD. Proton ((1) H) NMR spectroscopy revealed almost identical profiles between RIO and native LPD. The clinical mammography unit, 128-slice CT scanner and 3.0T MRI magnet were well adapted for the animal experiments. Combined use of RIO with DR, MRI, CT and histology enabled microangiography of the organs, 3D visualization of rat pancreas, validation of in vivo cardiac quantification of MI and cause determination of the rabbit death after coronary occlusion. RIO appeared as red droplets and vacuoles in vessels by frozen and paraffin sections. Image analysis showed the superiority of DR images, which provided better overall image quality (4.35 ± 0.49) for all analyzed liver vessel segments. MRI images revealed moderate to good overall image quality ratings (3.45 ± 0.52). Comparing the signal intensities of vessel and liver with different MRI sequences, all P values were <0.01.
CONCLUSIONS: RIO proved to be a multifunctional contrast dye, which could be applied as an imaging biomarker for tissue vascularity or blood perfusion, for visualization of organ anatomy and for ex vivo validation of in vivo animal experiments.