(-)-, (+)- and (±)- NBP (purity>99%) were supplied by Professor Jing-Hua Yang of the Department of Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences. The drugs were prepared into emulsion with 0.5% Tween-80 before being used.

The experimental protocol was approved by the local committee on ethics of animal experimentation. All animal procedures were performed on adult male Wistar rats weighing 270 to 300 g. Animals were kept under standard conditions with free access to food and water before and after surgery. Focal cerebral ischemia was induced by intraluminal middle cerebral artery (MCA) blockade with a nylon suture, as previously described (₁₁). Briefly, rats were anesthetized with 10% chloral hydrate (350mg/kg), the rectal temperature were maintained in the range of 36.5 to 37.5?. The bifurcation of the common carotid artery was exposed, the external carotid artery was dissected and coagulated distally, and the internal carotid artery was isolated and separated from the vagus nerve. A 40mm length of monofilament nylon suture (?0.26mm) was introduced into the internal carotid artery through the stump of the external carotid artery and gently advanced for a distance of 20 mm from the common carotid artery bifurcation to block the origin of the MCA. The MCA blood flow was restored by the withdrawal of the nylon suture 2 hours after occlusion. Sham operations were performed using the same surgical procedures except that no suture was inserted. Animals were divided into seven groups (n=10 for each): 1) sham-operated animals; 2) vehicle treated animals: only 0.5% Tween-80 was administrated; 3 and 4) (-)- NBP 5 and 10 mg/kg treated animals; 5 and 6) (+)- NBP 5 and 10 mg/kg treated animals; 7) (±)- NBP 20 mg/kg treated animals. (-)-, (+)- and (±)- NBP were given i.p. 10 min after ischemia.

The evaluation of neurological deficits was carried out at 24 hours after reperfusion using the neurological grading system (grading of 0 to 3) developed by Bederson et al. (₁₂). The rater was naive to the treatment protocol and to the identity of the groups.

At 24 hours after reperfusion, animals were anesthetized with 10% chloral hydrate (350 mg/kg i.p.) and perfused with 0.1 M phosphate-buffered saline (PBS) (pH 7.4) followed by 4% paraformaldehyde-buffered solution. The paraffin–embedded coronal sections (10µm thick) at the level of the caudate putamen that showed typical infarction were selected and processed for TUNEL staining, according to the method of Gavrieli et al. (₁₃). Briefly, brain sections were placed in terminal deoxynucleotidyl transferase (TdT) buffer for 15 min, followed by reaction with TdT enzyme and biotinylated 11-dUTP at 37? for 60 min. Avidin-horseradish peroxidase solution was applied to the sections, and the staining was visualized with diaminobenzidine. Slides were then counterstained with methyl green, dehydrated, and mounted.

At 24 hours after reperfusion, animals were anesthetized with 10% chloral hydrate (350 mg/kg i.p.) and decapitated. The brains ipsilateral to the ischemic hemisphere were rapidly removed and homogenized in lysis buffer containing 0.5% sodium dodecyl sulfate, 10 mM Tris-HCl, pH 8.0, 0.1 M EDTA, 100 µg.ml-1 proteinase K (Merck) and incubated in the same buffer for 3 hours at 50?. After digestion, samples were treated with 20 µg.ml-1 DNA-free RNase (Sigma) for 1 hours at 37?. Then DNA was extracted with equal volumes of phenol and phenol-chloroform-isoamyl alcohol (25:24:1), and precipitated for 2 hours in 3 M NaAc, pH 5.2 in 100% ethanol at -20?. The DNA was washed with 70% ethanol, air dried, and resuspended in Tris-EDTA buffer. Gel electrophoresis for detecting DNA laddering was performed with the TACSTM Apoptotic DNA Laddering Kit (Trevigen), as previously described (₁₄). First, the samples were reacted with Klenow enzyme (Trevigen) and dNTP (Trevigen) in 1×Klenow buffer (Trevigen). Then, samples were subjected to electrophoresis on a 1.5% agarose gel. After that, the gel was washed with 0.25 M HCl, 0.4 M NaOH or 0.8 M NaCl, and 0.5 M Tris, pH 7.5. Later on, DNA was transferred to a nylon membrane. The membrane was first blocked by 5% powdered milk (Sigma) in PBS, then incubated with streptavidin-horseradish peroxidase conjugate (Trevigen). Finally, the bands were visualized by chemiluminescence method, and the films were exposed to X-ray film.

At 3, 6, 12, 24 and 72 hours after reperfusion, animals were anesthetized with 10% chloral hydrate (350 mg/kg i.p.) and ipsilateral hemispheric brains were obtained. Homogenization was performed in ice-cold buffer [50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5 µg.ml-1 aprotinin], and centrifuged at 12000×g at 4? for 30 min. Total proteins present in the supernatant were then collected.

Protein extraction of both the mitochondrial and the cytosolic fractions was performed as described (14). Briefly, approx. 300 mg of sham-operated brain or ischemic brain was cut into pieces and gently homogenized by being dounced 30 times in a glass tissue grinder in 7 volumes of cold suspension buffer (20 mM HEPES-KOH, pH 7.5, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 2 mM EDTA, 0.1 mM PMSF, 2 µg.ml-1 aprotinin and 10 µg.ml-1 leupeptin). The homogenates were centrifuged at 800×g for ₁₅ min at 4? and then at 8000×g for 20 min at 4?. The 8000×g pellets were used to obtain the mitochondrial fraction. The supernatant was further centrifuged at 100000×g for 60 min at 4?. Protein concentrations were determined by the Lowry method.

Ten mg of each protein sample was electrophoresed through a 15% sodium dodecyl sulfate polyacrylamide gel. The primary antibodies were either a mouse monoclonal antibody directed against cytochrome c (7H8.2C12, Pharmingen, 1:1000 final dilution) or a rabbit polyclonal antibody against caspase-3 (Santa Cruz Biotechnology, 1:200 final dilution), which recognized the 32 kDa unprocessed procaspase-3 and the 17 kDa, but not the 12 kDa subunits of the active caspase-3. Western blots were performed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG using enhanced chemiluminescence Western blotting detection reagents (Santa Cruz Biotechnology). The optical density of the corresponding Western blot band was measured using Kodak ID Image. Data were calculated as % of optical density levels of the vehicle group. Western blot analysis of b-actin was also performed.

Rats were reanesthetized by 10% chloral hydrate (350 mg/kg i.p.) at 24 hours after reperfusion. After transcardiac perfusion, brains were cryoprotected in 25% sucrose in PBS for over 48 hours at 4?, and coronal brain sections (20µm thick) were cut on a cryostat (Slee Medical Equipment Ltd, UK) and thaw-mounted onto 3-amino- propyltriethoxy saline coated slides. An ABC Kit (Zhong Shan Biotechnology, Beijing) was used to localize the primary antibody and a Diaminobenzidine Kit (Zhong Shan Biotechnology, Beijing) was used to visualize the catalyzed peroxidase-reaction product. Sections were then counterstained with methyl green, dehydrated, cleared, and coverslipped. The mouse monoclonal antibody against rat cytochrome c (7H8.2C12, Pharmingen) was used at a 1:500 dilution and the rabbit polyclonal antibody against caspase-3 (Santa Cruz Biotechnology) was used at a 1: 100 dilution. As a negative control, sections were incubated without primary antibodies.

As shown in Figure 2, (-)- NBP at the doses of 5 and 10 mg/kg significantly inhibited the neurological deficit as compared to vehicle group (n=10, P<0.05). In contrast, no significant effect was observed in (+)- NBP (5, 10 mg/kg) treated rats (n=10, P>0.05). Additionally, (±)- NBP 20 mg/kg led to less reduction in the grading of neurological deficit than (-)- NBP 10 mg/kg did.

Figure 2

Figure 2: Effects of chiral 3-n-butylphthalide (NBP) on neurological deficits in MCAO rats (mean of n=10±s.d)

* P<0.05, ** P<0.01, *** P<0.001 vs vehicle group.

According to our previous experiments (unpublished observations), the time-point of 24 hours after reperfusion was selected to study the effects of chiral NBP on DNA fragmentation, which were characterized by TUNEL analysis and DNA laddering.

Figure 3

Figure 3: (Top) TUNEL analysis of neuronal cells in MCAO rats treated with chiral 3-n-butylphthalide (NBP). (Bottom) Representative photomicrographs of cytochrome c (a to d) and caspase-3 (e to h) immunostaining in MCAO rats treated with chiral NBP.

Figure 4

Bar = 15 µm.

TUNEL staining has been used extensively to identify cells with nuclear DNA fragmentation. Cells were scored as apoptosis when they were TUNEL-positive (brown staining) and showed nuclear chromatin condensation (arrows in Figure 3 top, b). In each hemispheric section of the sham-operated rats and in the contralateral hemisphere of ischemic rats, cells were almost negative for TUNEL staining, with only zero to three TUNEL-positive cells. These cells with nuclei not stained with TUNEL (TUNEL-negative, green-colored) were visualized by counterstaining with methyl green (Figure 3 top, a). In contrast, in the ipsilateral hemisphere of the ischemic brain in the vehicle group, a large number of TUNEL-positive cells, which were mainly located in the parietal cortex (265±16) and in the inner boundary of the caudate putamen (387±21), were seen at 24 hours after reperfusion (Figure 3 top, b, Figure 4). (-)- NBP (5, 10 mg/kg) dose-dependently reduced the number of TUNEL-positive cells compared with the vehicle group (Figure 3 top, c, Figure 4). The higher dose (10 mg/kg) reduced the number of TUNEL-positive cells to 140±9 (n=3) of the parietal cortex and to 275±14 of the caudate putamen, and the lower dose (5 mg/kg) led to a reduction of 225±10 in the parietal cortex (Figure 4). However, only the higher dose of (+)- NBP (10 mg/kg) showed slight inhibitory effect (Figure 4). Surprisingly, the inhibitory effect of (±)- NBP 20 mg/kg was significantly less than that of (-)- NBP 10 mg/kg (Figure 4, P<0.05).

Figure 5

Figure 4: Quantitative evaluation of the effects of chiral 3-n-butylphthalide (NBP) on the number of apoptotic cells in MCAO rats (mean of n=3±s.d).

* P<0.05, ** P<0.01, *** P<0.001 vs vehicle group. (a) P<0.05, (b) P<0.01 vs (-)- NBP (10 mg/kg) treated rats.

No DNA laddering was seen in sham-operated rats (Figure 5, lane 1), whereas a significant amount of DNA laddering was detected in vehicle treated rats (n=4) (Figure 5, lane 2) at 24 hours after reperfusion. DNA laddering was markedly inhibited in dose-related fashion in (-)- NBP (5, 10 mg/kg) treated rats (n=4) (Figure 5, lane 3, 4) compared with vehicle group. (-)- NBP 10 mg/kg almost completely inhibited DNA laddering, whereas (+)- NBP 10 mg/kg (Figure 5, lane 6) showed weak effect. Interestingly, the inhibitory effect of (±)- NBP (20 mg/kg) (Figure 5, lane 7) seemed to be in the range between that of (-)- NBP (10 mg/kg) and (+)- NBP (10 mg/kg).

Figure 6

Figure 5: Effects of chiral 3-n-butylphthalide (NBP) on DNA laddering induced by MCAO in rat brain.

Western blot analysis

Cytochrome c immunoreactivity was obvious as a unique 15kDa band detected by Western blot analysis. When a strong immunostaining was present in mitochondrial fractions of brain from sham-operated rats, no band was seen in the cytosolic fractions (Figure 6a to 6b, lane 1). Figure 6a shows the time-dependent release of cytochrome c during reperfusion after 2 hours of MCAO. The immunoreactivity of the mitochondrial fractions was decreased as early as 3 hours after reperfusion in ischemic brain, with a corresponding increase in cytosolic fractions (Figure 6a, lane 2). The release of cytochrome c was found to be max. at 24 hours after reperfusion (Figure 6a, lane 5) and the effects of chiral NBP were therefore evaluated at this time-point in subsequent studies. (-)- NBP (5, 10 mg/kg) (Figure 6b, lane 3, 4) dose-dependently inhibited the distributional change of cytochrome c, whereas (+)- NBP (5, 10 mg/kg) (Figure 6b, lane5, 6) showed no significant effect (Figure 6c). Surprisingly, (±)- NBP 20 mg/kg (Figure 6b, lane 7) showed less effect than (-)- NBP 10 mg/kg did (Figure 6c). On the other hand, a consistent amount of ß-actin immunoreactivity was seen in the bottom panel, suggesting that the amount of the loaded protein was consistent (Figure 6a to 6b).

Figure 7

Figure 6: Western blot analysis of cytochrome c in cytosolic and mitochondrial fractions from MCAO rats.

Each column represents mean ± s.d. (n=3). * P<0.05, ** P<0.01 and ***P<0.001 vs vehicle treated rats, ### P<0.001 vs sham-operated rats.

Immunohistochemistry

There was no immunoreactivity in the sections of sham-operated rats (Figure 3 bottom, a) or in the negative control sections (Figure 3 bottom, b) which were treated without primary antibody. The absence of immunoreactivity is considered to be caused by thorough fixation of the brain by paraformaldehyde, which prevents the antibody from reaching the mitochondrial intermembrane space, but not the cytosol (14). The massive increase of cytoplasmic cytochrome c immunoreactivity in vehicle treated rats (Figure 3 bottom, c) at 24 hours after reperfusion demonstrated the release of cytochrome c into the cytosol. The population of immunoreactive cells as well as the intensity of cytoplasmic immunostaining was significantly reduced in (-)- NBP (5, 10 mg/kg) treated rats (Figure 3 bottom, d, Table 1), whereas the higher dose of (+)- NBP (10 mg/kg) showed a weaker effect, and the inhibitory effect of (±)- NBP (20 mg/kg) was in the range between that of (-)- NBP (10 mg/kg) and (+)- NBP (10 mg/kg), a profile similar to that obtained by Western blot analysis (Table 1). Moreover, rats treated with (-)- NBP showed less immunostaining of the background, and some cells were accompanied by a long, axon-like structure protruding from the cytosol (arrows in Figure 3 bottom, d). This might reflect the improving effect of (-)- NBP on the integrity of cells.

Figure 8

Table 1: Effects of chiral 3-n-butylphthalide (NBP) on the expression of cytoplasmic cytochrome c protein and caspase-3 protein as measured by immunohistochemistry in MCAO rats.

-, no expression; +, weakly detectable expression; ++, prominent expression; +++, intense expression (n=3).

Western blot analysis

Normally, caspase-3 protease is synthesized in cells as an inactive precursor (32 kDa), which is cleaved into a small prodomain and two subunits of 17 (p17) and 12 kDa (p12) when activated. The antibody we used recognizes both the precursor and the p17 but not the p12 in the brain protein extracts (Figure 7). Brains from sham-operated rats showed high levels of precursor but none or very low levels of p17 subunit (Figure 7a to 7b, lane 1). The precursor levels decreased and the p17 subunit appeared at 3 hours after reperfusion (Figure 7a, lane 2) and peaked at 24 hours after reperfusion (Figure 7a, lane 5). Therefore, the effects of chiral NBP were investigated at 24 hours after reperfusion (Figure 7b to 7c). Similar to the effects on cytochrome c, (-)- NBP (5, 10 mg/kg) (Figure 7b, lane 3, 4) inhibited ischemia induced activation of caspase-3 in a dose-dependent manner, whereas (+)- NBP (10 mg/kg) (Figure 7b, lane 6) showed a weaker effect (Figure 7c). The inhibitory effect of (±)- NBP (20 mg/kg) (Figure 7b, lane 7) was shown to be in the range between that of (-)- NBP (10 mg/kg) and (+)- NBP (10 mg/kg) (Figure 7c).

Figure 9

Figure 7: Western blot analysis of the procaspase-3 (32 kDa) and its active fragment (17 kDa) in MCAO rats

Each column represents mean ± s.d. (n=3). * P<0.05, ** P<0.01 and ***P<0.001 vs vehicle treated rats, ###P<0.001 vs vehicle treated (sham-operated) rats.

Immunohistochemistry

This work was supported by the State Science and Technology Commission grant (No. 94-ZD-01) and by the National Natural Science Foundation of China (No. 29790122). The authors thank Professor Jing-Hua Yang (Department of Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences) for providing (-)-, (+)- and (±)- NBP and Professor Zhen-Yu Song for reading the manuscript. Professor Yi-Pu Feng gave us valuable directions in the experiments.