Effects of Dl-3-n-butylphthalide on cognitive functions and blood-brain barrier in chronic cerebral hypoperfusion rats

Abstract

Vascular cognitive impairment (VCI) has been one of the major types of cognitive impairment. Blood-brain barrier damage plays an essential part in the pathogenesis of VCI. At present, the treatment of VCI is mainly focused on prevention, with no drug clinically approved for the treatment of VCI. This study aimed to investigate the effects of DL-3-n-butylphthalide (NBP) on VCI rats. A modified bilateral common carotid artery occlusion (mBCCAO) model was applied to mimic VCI. The feasibility of the mBCCAO model was verified by laser Doppler, ¹³N-Ammonia-Positron Emission Computed Tomography (PET), and Morris Water Maze. Subsequently, the Morris water maze experiment, Evans blue staining, and western blot of tight junction protein were performed to evaluate the effect of different doses of NBP (40 mg/kg, 80 mg/kg) on the improvement of cognitive impairment and BBB disruption induced by mBCCAO. Immunofluorescence was employed to examine the changes in pericyte coverage in the mBCCAO model and the effect of NBP on pericyte coverage was preliminarily explored. mBCCAO surgery led to obvious cognitive impairment and the decrease of whole cerebral blood flow, among which the blood flow in the cortex, hippocampus and thalamus brain regions decreased more significantly. High-dose NBP (80 mg/kg) improved long-term cognitive function in mBCCAO rats, alleviated Evans blue leakage and reduced the loss of tight junction proteins (ZO-1, Claudin-5) in the early course of the disease, thereby exerting a protective effect on the blood-brain barrier. No significant changes in pericyte coverage were observed after mBCCAO. High-dose NBP improved cognitive function in mBCCAO rats. High-dose NBP protected the integrity of BBB by upregulating TJ protein expression, rather than regulating pericyte coverage ratio. NBP could be a potential drug for the treatment of VCI.

Keywords: Blood–brain barrier; Chronic cerebral hypoperfusion; Pericyte; Vascular cognitive impairment.

Fig. 1

Study design. (a) For verifying the effect of mBCCAO modeling, SD rats were randomly divided into two groups: sham group and model group. TTC staining was conducted on the day after mBCCAO. CBF change was monitored by laser Doppler flowmeter during the whole mBCCAO or sham surgery process and 1 week after surgery. Morris Water Maze experiment and ¹³N-Ammonia-PET scan were conducted 28 days after mBCCAO; (b) For evaluating the therapeutic effect of NBP on mBCCAO rats, SD rats were randomly divided into 4 groups: sham group, model group, low-dose butylphthalide group (L-NBP group) and high-dose butylphthalide group (H-NBP group). Cognitive function was evaluated by the Morris Water Maze experiment 28 days after surgery, Evans blue leakage experiment was conducted on 7 days and 28 days after mBCCAO to examine the BBB damage. Quantitative analysis of tight junction protein by Western blot on 7 days after surgery, as well as pericyte coverage by immunofluorescence on 3, 7 and 14 days, were conducted to explore the mechanism of NBP’s protective effect on mBCCAO rats

Fig. 2

CBF change after mBCCAO. (a) TTC staining, 24 h after modeling by mBCCAO; (b) schematic diagram of cerebral blood flow measured by laser Doppler (time bar = 10 min); (c) changes of CBF in the model group during operation and 1 w after BCCAO; (d) changes of CBF in the sham group during the same period. ***RCCAO and BCCAO group compared with the condition before the operation, P < 0.001. ##BCCAO group compared with the condition 1w after operation, P < 0.01. ns: indicates no statistical difference, n = 6 for each group. Values are expressed as ($\overline{x}$±SEM)

Fig. 3

The cognitive impairment after mBCCAO. (a) The spatial learning ability of the rats in each group was compared by evaluating their average escape latency to reach the target platform on every training day; (b) the total distance each group swam during the training period, which is also an indicator of spatial learning ability; (c) the average swimming speed of each group; (d) the representative movement trajectories of the two groups searching for the target platform; (e) the number of times rats in each group crossed the target platform (removed) on the last day of the spatial exploration experiment to test their spatial memory ability; (f) the percentage of time spent searching for the removed platform in the target quadrant for the two groups. n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no statistical difference. Values are expressed as ($\overline{x}$±SEM)

Fig. 4

Quantitative analysis of CBF in the whole brain and different brain regions in the sham and model group. (a, b) The pseudo-color maps were derived from ¹³N-Ammonia-PET imaging and showed representative whole-brain perfusion in the sham (a) and model group (b) respectively. The red signal in the pseudo-color map represents the hyperperfusion state, while blue or green represents the hypoperfusion state. The circles represent the interested areas selected for CBF measurement; (c-g) The bar graphs represent the quantitative results of CBF in the whole brain (c), hippocampus (d), thalamus (e), cerebellum (f), and different parts of the cortex (g) in the two rat groups. The model group (n = 3) compared with the sham group (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no statistical difference. Values are expressed as ($\overline{x}$±SEM)

Fig. 5

High-dose NBP improved cognitive function in CCH rats. (a) The spatial learning ability of rats in each group was reflected by comparing their average escape latency to the target platform; (b) the average swimming speed of each group on every training day; (c) the total swimming distance of each group during the training period; (d) the number of times rats crossing the target platform (removed) on the last day of spatial exploration experiment; (e) the percentage of time spent searching for the removed platform in the target quadrant for each group. n = 4–8, *P < 0.05, **P < 0.01, ***P < 0.001. ns indicates no statistical difference. Values are expressed as ($\overline{x}$±SEM)

Fig. 6

High-dose NBP ameliorated early BBB disruption in CCH rats. (a) EB leakage of rat brains in each group on day 7 and day 28 after surgery; (b) EB dye injection 2 h later, the eyes, ears, and upper and lower limbs of experimental rats turned blue; (c) EB leakage levels in brain tissue of rats in each group on day 7 and day 28 after surgery; (d) western blot representative bands of Claudin-5 and ZO-1; (e) quantitative analysis of Claudin-5 protein in each group; (f) quantitative analysis of ZO-1 protein in each group. For each group n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no statistical difference. Values are expressed as ($\overline{x}$±SEM)

Fig. 7

The changes of pericyte coverage in the cortex on days 3, 7, and 14 post-modeling. (a) The observation field was selected as the right motor cortex on the level near bregma; (b) Pericyte and vascular staining on sham, model, H-NBP rats on day3; (c) Quantitative analysis of the average ratio of PDGFRβ:CD31 on day3; (d) Pericyte and vascular staining on control, model, H-NBP rats on day7; (e) Quantitative analysis of the average ratio of PDGFRβ:CD31 on day7; (f) Quantitative analysis of the average ratio of PDGFRβ:CD31 on day14; (g) Pericyte and vascular staining on control, model, H-NBP rats on day14. For each group n = 3–6, ns indicates no statistical difference. Values are expressed as (x ® ± SEM). Scale bar: 100um