Effects of Dl-3-n-butylphthalide on Cerebral Ischemia Infarction in Rat Model by Mass Spectrometry Imaging

Dl-3-n-butylphthalide (NBP) is a drug that is used in the treatment of ischaemic stroke. However, to the best of our knowledge, there are no systematic studies investigating the effects of dl-3-n-butylphtalide on the brain metabolism of small molecules. In this study, we first investigated the effects of dl-3-n-butylphthalide on the spatial distribution of small molecules in the brains of rats with permanent middle cerebral artery occlusion (pMCAO) using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI–TOF–MS) imaging. After pMCAO modelling or a sham operation, rats were given four mg/kg of dl-3-n-butylphthalide through the caudal vein or saline once a day for nine days. The degree of neurological deficit in rats was evaluated using the modified neurological severity score (mNSS). MALDI–TOF–MS imaging was used to observe the content and distribution of small molecules related to metabolism during focal cerebral ischaemia. Multiple reaction monitoring (MRM) mode with liquid chromatography tandem mass spectrometry (LC–MS/MS) was used to verify the results obtained from MALDI–TOF–MS imaging. These small molecules were found to be involved in glucose metabolism, ATP metabolism, the glutamate–glutamine cycle, malate aspartate shuttle, oxidative stress, and inorganic ion homeostasis. Of the 13 metabolites identified by MALDI–TOF–MS imaging, seven compounds, ATP, ADP, AMP, GMP, N-acetylaspartic acid, ascorbic acid and glutathione, were further validated by LC–MS/MS. Taken together, these results indicate that dl-3-n-butylphthalide significantly improved ATP metabolism, level of antioxidants, and sodium-potassium ion balance in a rat model of pMCAO.


Haematoxylin-eosin (HE) staining
Three whole brains for each group were removed and snap frozen. The section was fixed in 4% paraformaldehyde, embedded in paraffin and cut into 6 serial slices of 10 μm for HE staining. The cerebral coronal sections with HE staining were scanned under 40x magnification with an automated microscope (Motic BA600) and photographed using the Motic DSAssistant Lite software, version 1.0.

Preparation of the matrix
To obtain 1,5-DAN hydrochloride, 39.5 mg of 1,5-DAN was dissolved in 500 μL 1 mol/L hydrochloride and 4 mL of distilled water by sonication. Next, 4.5 mL of ethanol was added to the matrix solution.

Preparation of the mixture standard solution
A mixture standard solution containing taurine, L-aspartate, L-glutamate, glutamine, N-acetylaspartate, ascorbic acid, citric acid, glutathione, AMP, ADP, ATP, GMP, and glucose at 50 μM each was prepared in water.

Semi-quantitative analysis of MALDI-TOF-MS
For MALDI-TOF-MS, regions of interest (ROIs) (Supplementary figure 3) were defined on both the lesioned side and normal side. The average peak heights of the 15 altered metabolites in each ROI, which represented the content of the molecules, were calculated using the Bruker Daltonics flexImaging 3.0 software. The ratios of the altered metabolites on the injured side to the normal side are shown in Supplementary Table 4A, which indicates a similar conclusion to the pictured observations. The average peak heights are shown in Supplementary Table 4B.

Intra-day and inter-day precision of LC-MS/MS
A standard solution containing 12 components was prepared with a 20 μg/mL concentration for each component. The solution was then added to the rat brain extract that was dried under nitrogen (the rat brain extract was prepared according to the method described in section 4.6 using a normal rat brain). The mixture was centrifuged, and the supernatant was divided into 5 samples. All samples were injected once per day for three days to measure the inter-day precision. The intra-day precision was calculated based on the results of the first day. The RSD values should be under 15% to meet the requirement of precision (Supplementary Table 5). Figure S1. Kaplan-Meier survival curves for the four groups. Sham: sham surgery group; pMCAO: pMCAO group; NBP: dl-3-n-butyphthalide treated group; UK: urinary kallidinogenase group. Data were analysed and plotted using IBM SPSS Statistics Version 20 software. * p < 0.001 pMCAO vs Sham. Sham: sham surgery group; pMCAO: pMCAO group; NBP: dl-3-n-butylphthalide. Data were presented as the mean ± SD, n = 3 per group. A one-way ANOVA was used to analyse the differences between groups. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. sham group; *P < 0.05, **p < 0.01, ***p < 0.001 vs. pMCAO group. Supplementary Figure S2. Representative images of the brain morphology revealed by HE staining. (A) sham surgery, (B) pMCAO, (C) dl-3n-butylphthalide treated group and (D) urinary kallidinogenase treated group. The striatum on the lesioned side was scanned at 40x and is shown on the right. Scale bar=2 mm for the full coronal section. Scale bar = 100 μm for microscopic observation.