Cerebral endothelial cells mediated enhancement of brain pericyte number and migration in oxygen-glucose deprivation involves the HIF-1α/PDGF-ß signaling.

The present study focused on whether hypoxia-inducible factor-1alpha (HIF-1α) and platelet-derived factor-beta (PDGF-ß) are involved in the crosstalk between brain microvascular endothelial cells (BMECs) and brain vascular pericytes (BVPs) under ischaemic-hypoxic conditions. Mono-cultures or co-cultures of BVPs and BMECs were made for the construction of the blood-brain barrier (BBB) model in vitro and then exposed to control and oxygen-glucose deprivation (OGD) conditions. BBB injury was determined by assessing the ability, apoptosis, and migration of BVPs and the transendothelial electrical resistance and horseradish peroxidase permeation of BMECs. Relative mRNA and protein levels of HIF-1α and PDGF-ß, as well as tight junction proteins ZO-1 and claudin-5 were analyzed by western blotting, reverse transcription quantitative PCR, and/or immunofluorescence staining. Dual-luciferase reporter assays assessed the relationship between PDGF-ß and HIF-1α. Co-culturing with BMECs alleviated OGD-induced reduction in BVP viability, elevation in BVP apoptosis, and repression in BVP migration. Co-culturing with BVPs protected against OGD-induced impairment on BMEC permeability. OGD-induced HIF-1α upregulation enhanced PDGF-ß expression in mono-cultured BMECs and co-cultured BMECs with BVPs. Knockdown of HIF-1α impaired the effect of BMECs on BVPs under OGD conditions, and PDGFR-ß silencing in BVPs blocked the crosstalk between BMECs and BVPs under OGD conditions. The crosstalk between BMECs and BVPs was implicated in OGD-induced BBB injury through the HIF-1α/PDGF-ß signaling.

Rectangular method: a modified technique for sampling the ischemic border zone in a rat model of transient middle cerebral artery occlusion.

To date, there have been three common methods for sampling the cerebral ischemic border zone in a rat model of transient middle cerebral artery occlusion (tMCAO): the "two o'clock method", the "diagonal method", and the "parallel line method". However, these methods have their own advantages and limitations. Here, we propose a modified technique (the "rectangular method") for sampling the ischemic border zone. A rat tMCAO model was prepared under the support of a compact small animal anesthesia machine. Cerebral blood flow was monitored by high-resolution laser Doppler to control the quality of modeling, and 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used for cerebral infarction location assessment. Superoxide dismutase 2 (SOD2), cysteinyl aspartate specific proteinase (caspase)-3, caspase-9, and heat shock protein 70 (HSP70) were used to verify the reliability and reproducibility of the rectangular method. The expression of biomarkers (SOD2, caspase-3, caspase-9, and HSP70) in the traditional (two o'clock method after TTC staining) and modified (rectangular method) groups were increased. There were no significant differences between the groups. The rectangular method proposed herein is based on a modification of the diagonal method and parallel line method, which could provide a directly observable infarct borderline and a sufficient sampling area for subsequent experimental operations regardless of the cerebral infarct location. The assessed biomarkers (SOD2, caspase-3, caspase-9, and HSP70) demonstrated the reliability and reproducibility of the rectangular method, which may facilitate inter-laboratory comparisons.

Rectangular method: a modified technique for sampling the ischemic border zone in a rat model of transient middle cerebral artery occlusion

To date, there have been three common methods for sampling the cerebral ischemic border zone in a rat model of transient middle cerebral artery occlusion (tMCAO): the "two o'clock method", the "diagonal method", and the "parallel line method". However, these methods have their own advantages and limitations. Here, we propose a modified technique (the "rectangular method") for sampling the ischemic border zone. A rat tMCAO model was prepared under the support of a compact small animal anesthesia machine. Cerebral blood flow was monitored by high-resolution laser Doppler to control the quality of modeling, and 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used for cerebral infarction location assessment. Superoxide dismutase 2 (SOD2), cysteinyl aspartate specific proteinase (caspase)-3, caspase-9, and heat shock protein 70 (HSP70) were used to verify the reliability and reproducibility of the rectangular method. The expression of biomarkers (SOD2, caspase-3, caspase-9, and HSP70) in the traditional (two o'clock method after TTC staining) and modified (rectangular method) groups were increased. There were no significant differences between the groups. The rectangular method proposed herein is based on a modification of the diagonal method and parallel line method, which could provide a directly observable infarct borderline and a sufficient sampling area for subsequent experimental operations regardless of the cerebral infarct location. The assessed biomarkers (SOD2, caspase-3, caspase-9, and HSP70) demonstrated the reliability and reproducibility of the rectangular method, which may facilitate inter-laboratory comparisons.