Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells.

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro. This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage.

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility adjustments. Conventionally, their contractility is gauged by observing morphological shifts and nearby capillary diameter changes under specific circumstances. Yet, post-tissue fixation, evaluating vitality and ensuing pericyte contractility of imaged brain pericytes becomes compromised. Similarly, genetically labeling brain pericytes falls short in distinguishing between viable and non-viable pericytes, particularly in neurologic conditions like subarachnoid hemorrhage (SAH), where our preliminary investigation validates brain pericyte demise. A reliable protocol has been devised to surmount these constraints, enabling simultaneous fluorescent tagging of both functional and non-functional brain pericytes in brain sections. This labeling method allows high-resolution confocal microscope visualization, concurrently marking the brain slice microvasculature. This innovative protocol offers a means to appraise brain pericyte contractility, its impact on capillary diameter, and pericyte structure. Investigating brain pericyte contractility within the SAH context yields insightful comprehension of its effects on cerebral microcirculation.

[The effect of simvastatin on osteoblastic bionomics of rats].

PURPOSE: To study the effect of simvastatin on osteoblast proliferation, differentiation and mineralization and to explore the mechanism of stimulation of bone formation by simvastatin. METHODS: Six 8-hour healthy SD rats were used for the experiment. In order to harvest vital osteoblast, this research modified the traditional tissue piece method to culture primary osteoblast of rat calvarias.The effect of simvastatin in different concentration(1.0micromol/L,0.5micromol/L,0.25micromol/L,0.125micromol/L) on osteoblast was measured using MTT to assay osteoblasts proliferation, alkaline phosphatase activity of osteoblasts was measured, the level of osteocalcin (OC) was detected, the capacity of mineralization was investigated by counting the mean mineralization area with Image-Pro Plus 6.0.The data was analyzed with SPSS 13.0 software package for one-way ANOVA. RESULTS: Compared with the control group, the osteoblasts proliferation was inhibited in the experimental groups in a dose-dependent manner. It showed that the alkaline phosphatase activity of osteoblast was increased in all experimental groups. The level of OC of osteoblasts showed a significant increase in all treatment groups. With regard to osteoblast capability of mineralization, the mineralization area was also increased, compared with the control group (P<0.05). CONCLUSION: Simvastatin inhabits the proliferation of osteoblasts in vitro, but promotes differentiation and mineralization of osteoblast.

Effects of soil amendments at a heavy loading rate associated with cover crops as green manures on the leaching of nutrients and heavy metals from a calcareous soil.

The potential risk of groundwater contamination by the excessive leaching of N, P and heavy metals from soils amended at heavy loading rates of biosolids, coal ash, N-viro soil (1:1 mixture of coal ash and biosolids), yard waste compost and co-compost (3:7 mixture of biosolids to yard wastes), and by soil incorporation of green manures of sunn hemp (Crotalaria juncea) and sorghum sudangrass (Sorghum bicolor x S. bicolor var. sudanense) was studied by collecting and analyzing leachates from pots of Krome very gravelly loam soil subjected to these treatments. The control consisted of Krome soil without any amendment. The loading rate was 205 g pot(-1) for each amendment (equivalent to 50 t ha(-1) of the dry weight), and the amounts of the cover crops incorporated into the soil in the pot were those that had been grown in it. A subtropical vegetable crop, okra (Abelmoschus esculentus L.), was grown after the soil amendments or cover crops had been incorporated into the soil. The results showed that the concentration of NO3-N in leachate from biosolids was significantly higher than in leachate from other treatments. The levels of heavy metals found in the leachates from all amended soils were so low, as to suggest these amendments may be used without risk of leaching dangerous amounts of these toxic elements. Nevertheless the level of heavy metals in leachate from coal ash amended soil was substantially greater than in leachates from the other treatments. The leguminous cover crop, sunn hemp, returned into the soil, increased the leachate NO3-N and inorganic P concentration significantly compared with the non-legume, sorghum sudangrass. The results suggest that at heavy loading rates of soil amendments, leaching of NO3- could be a significant concern by application of biosolids. Leaching of inorganic P can be increased significantly by both co-compost and biosolids, but decreased by coal ash and N-viro soil by virtue of improved adsorption. The leguminous cover crop, sunn hemp, when incorporated into the soil, can cause the concentration of NO3-N to increase by about 7 fold, and that of inorganic P by about 23% over the non-legume. Regarding the metals, biosolids, N-viro soil and coal ash significantly increased Ca and Mg concentrations in leachates. Copper concentration in leachate was increased by application of biosolids, while Fe concentration in leachates was increased by biosolids, coal ash and co-compost. The concentrations of Zn, Mo and Co in leachate were increased by application of coal ash. The concentrations of heavy metals in leachates were very low and unlikely to be harmful, although they were increased significantly by coal ash application.