ABSTRACT
AIM: To optimize the technique of intravenous injection of Evans blue and retinal preparations in mice, improving the accuracy and repeatability of staining experiment of retinal preparations.METHODS: C57BL/6 male mice were intravenous injected with 10g/L(1%)Evans Blue 0.3mL and circulated in vivo for 10 or 20min, and the eyes were removed after sacrificed and fixed in 4% paraformaldehyde for 20, 40 or 60min. When failure of intravenous injection, the experiment was remediated by intraperitoneal injection of 1% Evans Blue 0.3mL, circulated in vivo for 3h and fixed for 60min to observe morphology, distribution and leakage of the retinal vessels. Besides, we compared the morphology, distribution and leakage of the retinal vessels after intravenous injection with those after intraperitoneal injection to determine the optimal conditions for in vivo circulation time and retinal preparations.RESULTS: After intravenous injection, compared to the retinal vascular condition under 20min in vivo circulation time of Evans blue and 20 or 40min of fixation, with 10min of in vivo Evans blue circulation and 60min of fixation, the morphology of retinal vascular was more intact with less retinal vascular leakage, and the vascular branches are clear. When intravenous injection failed, remediated results from intraperitoneal injection showed that the morphology and distribution of retinal vessels were intact. There was no significant difference in morphology, distribution and leakage of the retinal vessels after 3h of intraperitoneal Evans blue circulation compared to 10min intravenous Evans blue circulation.CONCLUSION: This experiment optimizes the protocol, improves the accuracy and reproducibility of retinal preparations, and provides a reference for the study of related retinal vascular diseases.
ABSTRACT
Endothelial progenitor cells (EPCs) are precursor cells of endothelial cells. Signal molecules produced by ischemia and hypoxia can promote mobilization of bone marrow EPCs to peripheral circulation and formation of novel blood vessels in tissues that are damaged during heart attack. Naoxintong capsule (NXT) has the functions of promoting blood circulation, removing blood stasis, promoting the circulation of qi and relieving pain. The various components in NXT have protective effects on blood vessels and can effectively improve the symptoms of ischemia. However, its effect on EPCs is not clear. To study the intervention effect of NXT on mobilization and homing of peripheral blood EPCs, green fluorescent protein (GFP) transgenic mice were used for bone marrow transplantation (BMT) and then unilateral hind limb ischemia model (UHLI) were constructed. For BMT, wild-type ICR mice were irradiated by CS137 and then injected with 4×106 bone marrow cells isolated from GFP mice. The bone marrow reconstitution of recipients was assessed by quantification of GFP bone marrow-derived cells (BMDC) from transplanted mice 4 weeks after BMT. The UHLI model was duplicated by ligating femoral artery and divided into three groups: the model group, the NXT group (model+NXT) and the positive control group (model+simvastatin). Flow cytometry was used to detect the proportion of GFP positive cells and the peripheral blood EPCs levels at 1, 3, 7, 14 days before and after surgery. Ischemic tissue of gastrocnemius muscle was excised at 3 and 7 days after operation for immunofluorescence staining to detect the number of GFP+ cells. The bone marrow chimerism was achieved at day 28 after BMT. There was no significant difference in the percentage of GFP positive cells between BMT mice and GFP transgenic mice. NXT and simvastatin could significantly increase the number of peripheral blood EPCs 1,3 days after surgery. Three and seven days after operation, the number of homing EPCs was significantly higher in NXT group and positive control group than that in model group (P<0.001). In conclusion, NXT can obviously promote the mobilization and homing of EPCs.
ABSTRACT
Naoxintong capsule has beneficial effects for activating blood circulation, dispersing blood stasis and dredging collateral. It is widely used in the treatment of coronary heart disease, angina pectoris, stroke and cardiovascular disease. However, the pharmacodynamic basis and possible mechanism of its preventive effects are not clear. In this study, 10 male and 10 female C57BL/6 mice were used, and were randomly divided into the control group (saline) and Naoxintong group. Adaptively fed for 7 days in common conditions, mice were given Naoxintong capsule or saline for 3 days via intragastric administration. Serum was collected from 6 mice in each group 1 h after the last administration. Serum proteins were prepared to do two-dimensional gel electrophoresis. Then image analysis and mass spectrometry detection were carried out to screen and identify the differentially expressed proteins and make bioinformatics analysis. It was found that 24 differentially expressed proteins between Naoxintong group and control group. Compared with the control group, 12 proteins were increased, and 12 were decreased. The proteins were involved in apoptosis signal pathway and vascular endothelial growth factor signal transduction pathway, in which vasohibin-1 is a negative feedback regulation factor in angiogenesis. Western blot showed that the expression of vasohibin-1 in Naoxintong group was reduced, which is consistent with the result in two-dimensional electrophoresis. Serum proteins expression is different between Naoxintong and control groups. The targets of these differentially expressed proteins include endothelial cells, inflammatory cells and platelets. The changes on proteins showed that Naoxintong capsule may ameliorate coronary heart disease and ischemic cerebrovascular disease, and provide potential biological markers to prevent ischemic disease.