FTY720 Reduces Endothelial Cell Apoptosis and Remodels Neurovascular Unit after Experimental Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. A sequence of pathological processes occurred when there is TBI. Previous studies showed that sphingosine-1-phosphate receptor 1 (S1PR1) played a critical role in inflammatory response in the brain after TBI. Thus, the present study was designed to evaluate the effects of the S1PR1 modulator FTY720 on neurovascular unit (NVU) after experimental TBI in mice. The weight-drop TBI method was used to induce TBI. Western blot (WB) was performed to determine the levels of SIPR1, claudin-5 and occludin at different time points. FTY720 was intraperitoneally administered to mice after TBI was induced. The terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay was used to assess endothelial cell apoptosis. Immunofluorescence and WB were performed to measure the expression of tight junction proteins: claudin-5 and occludin. Evans blue (EB) permeability assay and brain water content were applied to evaluate the blood-brain barrier (BBB) permeability and brain edema. Immunohistochemistry was performed to assess the activation of astrocytes and microglia. The results showed that FTY720 administration reduced endothelial cell apoptosis and improved BBB permeability. FTY720 also attenuated astrocytes and microglia activation. Furthermore, treatment with FTY720 not only improved neurological function, but also increased the survival rate of mice significantly. These findings suggest that FTY720 administration restored the structure of the NVU after experimental TBI by decreasing endothelial cell apoptosis and attenuating the activation of astrocytes. Moreover, FTY720 might reduce inflammation in the brain by reducing the activation of microglia in TBI mice.

Keywords: FTY720; blood-brain barrier; endothelial cell; sphingosine-1-phosphate receptor 1; traumatic brain injury.

Figure 1

A. The structure of the neurovascular unit (NVU) in normal and injured brain. The NVU consists of endothelial cells, perivascular astrocytes, microglia, and neurons. After injury, the structure of NVU is severely disturbed, which includes the decreases of tight junction proteins, astrogliosis, pericyte loss, endothelial cell apoptosis. B. The timeline of animal experiments in the present study.

Figure 2

A. Brain samples for Western blot analysis were taken as illustrated. The tissues from the penumbra area surrounding the trauma tissue was collected. B-E. The protein levels of S1PR1, claudin-5 and occludin in the sham group and in TBI groups (3, 6, 12, 24 and 72 h) were detected by Western blot analysis. Then, the intensity of the blots was quantified and analyzed by image J. Data were represented as the means ± SD (n = 6 per group), *p < 0.05, **p < 0.01, ***p < 0.001 versus the sham group. F. FTY720 treatment decreased the endothelial cell apoptosis. Fluorescence colors: CD31 (red); TUNEL(green); Dapi(blue). Scale bar, 50 µm. CD31, TUNEL and Dapi triple stained cells represented the apoptotic endothelial cells. G. Quantification of apoptotic endothelial cells in different groups. Data are presented as mean ± SD (n = 3 per group), *p < 0.05, **p < 0.01, ***p < 0.001 versus the sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the TBI +vehicle group. Scale bar, 50 µm.

Figure 3

A-D. Protein levels of p-ERK, claudin-5 and occludin were measured by Western blot analysis (n = 6 per group). E-G. Changes and location of claudin-5 and occludin were also detected by immunofluorescence staining. Data are presented as mean ± SD (n = 3 per group), *p < 0.05, **p < 0.01, ***p < 0.001 versus the sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the TBI+ vehicle group. Scale bar, 50 µm.

Figure 4

A. The mice were perfused after EB injection. The brain tissue stained with EB could be found in the area near the contusion. B. Photometric analysis of EB dye content showed that the EB content was higher in the TBI and TBI + vehicle groups than that in the TBI + FTY720 group. C. The animals in the TBI and TBI + vehicle groups had higher brain water content than the sham group. With administration of FTY720, brain water content was decreased. Data are presented as mean ± SD (n = 6 per group), *p < 0.05, **p < 0.01, ***p < 0.001 versus the sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the TBI + vehicle group.

Figure 5

A. Altered astrocytic and microglial morphology. GFAP and IBA-1 positive cells were found by immunohistochemistry in four groups. B,C. Quantification analyses. Cell densities for GFAP and IBA-1 were shown. Data are presented as mean ± SD (n = 3 per group), *p < 0.05, **p < 0.01, ***p < 0.001 versus the sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus TBI + vehicle group. Scale bar, 50 µm. E. The impact of FTY720 on the neurological function. The neurological function was evaluated with mNSS, n = 6/group. Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 versus TBI + vehicle group; and p > 0.05 versus TBI + vehicle group. F. Survival analysis for each group, n = 20/group. At the end of the observation period, the mortality was 20% in the FTY720 group, 65% in the TBI group and 45% in the vehicle group. Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 versus TBI + vehicle group; and p > 0.05 versus TBI + vehicle group.