Immortalized Mouse Cerebellar Capillary Endothelial Cells (cerebEND)

TTFields Effects on the BBB Were Dependent on the Frequency, Intensity, and Duration of the Treatment

The majority of therapeutic agents for CNS disorders are unable to penetrate the BBB, and thus their clinical effectiveness is extremely limited. Salvador et al. determined the effects of the clinically approved TTFields for GBM on blood-brain barrier permeability and integrity, which could also enhance CNS drug delivery.

To understand how TTFields affect the blood-brain barrier (BBB), they examined mouse cerebellum-derived endothelial cells (cerebEND) under different TTFields frequencies. Normally, these cells grow in a parallel, elongated pattern. But with TTFields, they formed swirls instead (Fig. 1A). They stained tight junction proteins (claudin-5 and ZO-1) to see changes better. After TTFields treatment, cells lost their elongated shape and became irregular (Fig. 1B). All tested frequencies (100-300 kHz) changed cell shape, but 100 kHz had the most visible effect. So, they used 100 kHz for later experiments. They also tested different intensities (1.62, 0.97, and 0.76 V/cm RMS) to see if they affected BBB opening. At 0.76 V/cm RMS, cells looked normal but had frayed edges. At 0.97 V/cm RMS, cells deformed, losing their elongated shape. The most dramatic changes were at 1.62 V/cm RMS, where claudin-5 distribution was most disturbed (Fig. 1C). This shows that cell response depends on intensity. Next, they tested how long it takes for 100 kHz, 1.62 V/cm TTFields to be most effective. Cells were treated for 24, 48, and 72 hours. Disruption was seen after 24 hours, but the most significant changes were after 72 hours (Fig. 1D).

Alterations in Tight Junction Proteins Expression After Oxygen-Glucose Deprivation in Murine Brain Capillary Cerebellar Endothelial Cells (cerebEND)

Stroke is the second leading cause of death and contributes to neuronal damage by inducing BBB failure and microvascular disruption, which is associated with edema. Stevioside, and its metabolite isosteviol sodium (STVNA), has been previously shown to exert neuroprotective effects in cerebral ischemia, but its effects on the BBB are not known. Here, Rösing et al. determined the effect of STVNA on the BBB in an in vitro stroke model, using oxygen and glucose deprivation (OGD), in murine brain capillary cerebellar endothelial cells (cerebEND).

Tight junction proteins (TJPs) are crucial for the barrier properties of brain capillary endothelial cells in the BBB. When the BBB is damaged, such as during ischemia, TJP expression can change. To study this, cerebEND cells were subjected to oxygen-glucose deprivation (OGD) and then treated with various concentrations (0, 1, 5, 10, and 20 mg/l) of isosteviol sodium (STVNA) for 4 and 24 hours. Western blot analysis showed that claudin-5 expression increased after 4 hours of STVNA treatment, with 5 mg/l yielding the highest increase (Fig. 2A, B). However, after 24 hours of STVNA treatment, claudin-5 expression decreased (Fig. 2C, D). Similar trends were observed for occludin (Fig. 2G, H), with the highest increase after 4 hours of STVNA treatment at both 5 and 10 mg/l (Fig. 2E, F). Additionally, qRT-PCR analysis showed that 24 hours of STVNA treatment post-OGD increased claudin-5 mRNA expression for all tested concentrations (Fig. 3).