Effect of Tetramethylpyrazine on Neuroplasticity after Transient Focal Cerebral Ischemia Reperfusion in Rats

2.1. Animals

Eight-week-old male Sprague Dawley (SD) rats weighing 200–250 g (purchased from Experimental Animal Center of Wuhan University) were used for this experiment. The rats were acclimated for 3 or more days before the start of any experiments. They were housed in a controlled environment (four animals per cage, 55 ± 5% relative humidity, 22°C, 12 : 12 h light/dark cycle) and provided with free access to food and water. All experimental procedures involving animals were conducted in strict accordance with the guidelines approved by the Animal Care and Use Committee of Wuhan University Medical School. We made all efforts to minimize the number of animals used and their suffering.

2.2. Establishment of Model

Transient focal cerebral ischemia was induced by MCAO using the intraluminal filament technique with some modifications [16]. The rats were anesthetized with sodium pentobarbital (50 mg/kg) intraperitoneally, and after a median incision of the neck skin, the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully isolated. The right middle cerebral artery (MCA) was occluded with a monofilament nylon filament (Beijing Cinonteh Biotech Co, Beijing, China) by inserting it through the right CCA and gently advancing into the ICA up to a point approximately 17 mm distal to the bifurcation of the CCA. The filament was fixed in place, and the animal was allowed to recover from anesthesia. Reperfusion was performed by withdrawing the filament by about 10 mm after 120 min without anesthesia. A heating pad was used to maintain a rectal temperature of 37.0 ± 0.5°C during the surgical procedure. Sham-operated rats underwent the same procedure without inserting a nylon filament.

2.3. Grouping and Administration

The animals were randomly assigned into 3 groups: sham group, model group, and TMP group (n = 10 per group). In the TMP group, animals received 20 mg/kg TMP (Aladdin Chemistry Co, Shanghai, China) intraperitoneally immediately after reperfusion. In the sham and control groups, animals were served with injection of the same volume of saline at the same time. All injections were conducted through intraperitoneal injection daily and in volume of 5 ml/kg until the day before sacrificing. The animals were sacrificed at 3, 7, 14, and 28 d after MCAO for immunohistochemistry and 28 d for TEM. Figure 3 shows a brief flowchart of our study.

2.4. Neurological Functional Test

A modified neurological severity score (mNSS) test [17] was employed to evaluated neurological function (n = 10). It was performed at 3 d, 7 d, 14 d, and 28 d after MCAO in a blind fashion by another investigator. MNSS is a composite of motor, sensory, reflex, and balance tests and graded on a scale of 0–18 (normal score 0, maximal deficit score 18). A higher score means more severe injury.

2.5. Immunohistochemistry

At 3 d, 7 d, 14 d, and 28 d after MCAO, the rats in each group (n = 6) were anesthetized and perfused transcardially with 150 ml of 0.9% saline and 150 ml of 4% paraformaldehyde. The brain was removed and postfixed in 4% paraformaldehyde for 24 h at room temperature. Thereafter, the brain was cut into 4 mm thick coronal blocks (bregma: −2 to +2 mm). After embedded by paraffin, the sections of 6 μm were mounted onto the polylysine-coated slides. The immunohistochemical staining was performed using the streptavidin-peroxidase method [18] as follows: the tissue sections were dewaxed with xylene and rehydrated in ethanol. Then, the specimens were incubated in endogenous peroxidase blocking solution (Maixin Technology Co, Fuzhou, Fujian, China) for 10 min. After incubated with normal rabbit serum (Maixin Technology Co, Fuzhou, Fujian, China), the mouse anti-SYP primary antibody (1 : 200, Boster, Wuhan, Hubei, China) and mouse anti-GAP-43 primary antibody (1 : 200, Boster, Wuhan, Hubei, China) were added and incubated, respectively, with samples at 4°C overnight. A biotin-conjugated secondary antibody (Maixin Technology Co, Fuzhou, Fujian, China) was added to the samples and incubated at room temperature for 15 min. Then, each section was incubated with HRP-streptavidin-peroxidase (Maixin Technology Co, Fuzhou, China) for 15 min. Next, each section was treated with 3,3′-diaminobenzidine (DBA) and counterstained with hematoxylin. Between every procedure, the sections were rinsed with phosphate-buffered saline (PBS, pH = 7.4) 3 times for 3 min. Finally, the sections were dehydrated and cover-slipped. Negative controls were established by replacing the primary antibody with PBS and normal rabbit serum.

Another investigator evaluated immunohistochemical results blindly. Six randomly selected sections (three for GAP-43 and three for SYP) of each subject were used for statistic analysis. 5 arbitrary fields (40 × objective) from each section in IP of hemisphere with the lesion were randomly captured using a digital camera. Image Pro Plus 6.0 (IPP 6.0, Media Cybernetics Inc, Bethesda, MD, USA) was used to measure integral optical density (IOD) of every photographs.

2.6. Transmission Electron Microscopy

At 28 d after MCAO, the rats in each group (n = 4) were anesthetized and perfused transcardially with 250 ml 4% paraformaldehyde. 1 mm × 1 mm × 1 mm tissue samples were collected from parietal cortex of IP as quickly as possible (Figure 4(a)) and fixed in 2.5% phosphate-buffered glutaraldehyde for 24 h. Then, the samples were prepared for TEM routinely with some modifications [19]: (1) the samples were rinsed three times with 0.1 M PBS and postfixed in 1% OsO₄ for 2 h in 4°C; (2) after rinsed three times with 0.1 M PBS, they were dehydrated with ethanol and acetone in a graded series; (3) they were embedded in a 1 : 1 mixture of Epon 812 and acetone for 30 min; (4) the blocks were placed in 37°C for 24 h and then 60°C for 48 h; (5) the ultrathin sections (approximately 60 nm thick) were cut using an ultramicrotome (LKB-V, LKB Produkter AB, Sweden) and stained with uranyl acetate and lead citrate for 10 min each.

The sections were viewed at × 10000 under a HT-7700 TEM (Hitachi, Japan). In this study, the ultrastructures of synapses were evaluated using IPP 6.0 program. The parameters contained curvature of synaptic interface [20], thickness of postsynaptic density (PSD) [21], width of synaptic cleft (using multipoint average method), and length of the active zone [21] (Figures 4(b) and 4(c)). Totally, 40 micrographs (10 per animal) of each group were taken for measurement.

2.7. Statistical Analysis

All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by using the GraphPad Prism 8.3.0 (GraphPad Software, La Jolla, CA, USA). Immunohistochemistry data were analyzed using linear mixed-effects analyses followed by Tukey's post hoc comparisons. The data of mNSS were also analyzed using linear mixed-effects analyses while followed by Sidak's post hoc comparisons (model vs. TMP). The Greenhouse–Geisser correction was applied if the assumptions of sphericity were violated. TEM data were analyzed using one-way ANOVA followed by Tukey's post hoc comparisons. Correlation analysis between mNSS and other data was evaluated by Pearson's correlation test. A value of P value below 0.05 was considered statistically significant.

3.1. Effect of TMP on Neurological Function Recovery

A 18-point mNSS was used to measure the animals' neurological outcome following MCAO (n = 10 in every group). All rats in the sham group show 0 score without any neurological function impairment. Linear mixed-effects analyses indicated that there was a significant effect of time (F_3, 54 = 105.67, P < 0.001) and intervention (F_1, 18 = 10.01, P=0.005) on mNSS but no interaction between them (F_3, 54 = 1.70, P=0.177). Figure 5 shows that the model group and TMP group improved their neurological performance overtime, which displayed a declining curve of mNSS scores. According to the results of multiple comparisons, TMP significantly improved neurological function as evidenced by lower mNSS at 14 d (P=0.008) and 28 d (P=0.013) after MCAO.

3.2. Effect of TMP on Expression of SYP

In this study, IOD values were applied to indicate the expression of SYP and GAP-43. We obtained 15 IODs (5 fields × 3 sections), and the mean value was used for analysis (n = 6 in every group). There was a significant effect of time (F_3, 60 = 37.47, P < 0.001) and intervention (F_{2, 60} = 85.71, P < 0.001) on expression of SYP in IP; moreover, a significant interaction effect between time and intervention existed (F_{6, 60} = 7.56, P < 0.001). In the sham group, IOD values of SYP immunohistochemical staining were maintained at a stable level (3 d vs. 7 d vs. 14 d vs. 28 d, all P > 0.05 compared to each other). The expression of SYP in the model group was significantly higher than that of the sham group at 7 d (P=0.020), 14 d (P < 0.001), and 28 d (P < 0.001) after MCAO. Furthermore, SYP level was upregulated by TMP treatment at 7 d (P=0.016), 14 d (P < 0.001), and 28 d (P=0.002) after MCAO compared with the model group. Figure 6 shows the SYP levels of three groups.

3.3. Effect of TMP on Expression of GAP-43

The expression of GAP-43 was significantly affected by time (F_{1.78, 26.74} = 317.21, P < 0.001) and intervention (F_{2, 15} = 390.85, P < 0.001), and there was significant interaction (F_{6, 45} = 80.98, P < 0.001). Only weak GAP-43 immunostaining was observed in the sham group. In the control group, GAP-43 of IP appeared a sharply increase from 3 d to 7 d and reached the peak at 7 d; thereafter, the GAP-43 levels decreased gradually from 14 d to 28 d and still significantly was higher than that of the sham group (P < 0.001). In the TMP group, a similar pattern of time course was observed. However, TMP seemly could not regulate the GAP-43 levels at all time points (P > 0.05 compared to the model group). Figure 7 shows the GAP-43 levels of three groups.

3.4. Effect of TMP on Changes in Synaptic Ultrastructures

4 rats were sacrificed at 28 d after MCAO, and totally, 40 micrographs (10 per animal) of each group were taken for measurement. One-way ANOVA revealed that there were significant differences between three groups in curvature of synaptic interface (F2, 117 = 5.53, P=0.005), thickness of PSD (F2, 117 = 10.68, P < 0.001), and width of synaptic cleft (F2, 117 = 17.76, P < 0.001), whereas there is no difference in length of active zone (F2, 117 = 0.56, P=0.573). Compared with the sham group, the parameters of model group were partially changed with a less curved synaptic interface (P=0.007), a thinner PSD (P < 0.001), and a wider synaptic cleft (P < 0.001) at 28 d after MCAO. Some of these parameters could be modified by TMP to some extent. To be specific, synaptic interface curvature (P=0.029) and thickness of PSD (P=0.041) of TMP group were significantly higher compared with the sham group. Figure 8 shows the synaptic ultrastructural parameters of three groups.

3.5. Correlation Analysis

Pearson's correlation test showed that mNSS significantly correlated with SYP (r = −0.8310, P < 0.001), curvature of synaptic interface (r = −0.8296, P=0.011), thickness of PSD (r = −0.8323, P=0.010), and width of synaptic cleft (r = −0.7462, P=0.034) at 28 d after MCAO (Figures 9 and 10). However, there were no significant correlation between mNSS and GAP-43 (r = −0.2289, P=0.474) and length of the active zone (r = −0.3455, P=0.402) at 28 d after MCAO (Figures 9 and 10).