Tongxinluo promotes axonal plasticity and functional recovery after stroke

Abstract

Background: The aim of this study was to investigate the neural plasticity in contralesional cortex and the effects of tongxinluo (TXL) in cerebral ischemic rats.

Methodology: We used stroke-prone renovascular hypertensive (RHRSP) cerebral ischemia rat models to study the effect of TXL and the underlying mechanisms. We performed foot-fault and beam-walking tests to evaluate the motor function of rats after cortical infarction. Biotinylated dextran amine (BDA) was used to track axonal sprouting and neural connections.

Results: TXL enhanced the recovery of motor function in cerebral infarction rats. TXL increased axonal sprouting in the peri-infarcted area but not in the corpus callosum, indicating in situ origination instead of crossing between cortical hemispheres through the corpus callosum. TXL promoted the sprouting of corticospinal axons into the denervated side of spinal gray matter. The synaptophysin (SYN)-positive intensity in the peri-infarcted area of TXL-treated group was greater than that in the vehicle group. We observed co-localization of SYN with BDA-positive fibers in the denervated spinal cord gray matter in the TXL group, suggesting that axonal remodeling and synaptic connections were promoted by TXL.

Conclusion: TXL may promote the recovery of neurological function by promoting the axonal remodeling and synapse formation of motor neuronal fibers after focal cortical infarction in hypertensive rats.

Keywords: axonal remodeling; functional recovery; biotinylated dextran amines; distal middle cerebral artery occlusion; tongxinluo.

Figure 1

The experimental design and overview of the cerebral ischemia model. (a) Time line of the experiments. The time-points for pre-training of rats, dMCAO surgery, TXL or vehicle application, BDA injection, and sacrifice of rats are shown in red. Blue pointers indicate the time points for behavioral tests. (b) Schematic diagrams showing the locations of BDA injection and ischemic area in transverse section (left panel) and coronal section (right panel). Abbreviations: dMCAO, distal middle cerebral artery occlusion; BDA, biotinylated dextran amines.

Figure 2

Results of the foot-fault and beam-walking behavioral tests. (a) Foot-fault test: percentage of wrong footsteps (out of total footsteps) on days −1, 3, 7, 14, and 28 after dMCAO in the vehicle and TXL groups. On postoperative day 28, the wrong footstep rate of TXL groups (0.0305 ± 0.012) was lower than that of vehicle groups (0.0523 ± 0.0284). (b) Beam-walking test: the score of muscle strength degree on days −1, 3, 7, 14, and 28 after dMCAO in the vehicle and TXL groups. On postoperative day 28, the scores of TXL groups (5 ± 0.76) were higher than that of vehicle groups (4 ± 0.89). Data expressed as mean ± standard deviation. *P < 0.05 on ANOVA Tukey post hoc test. n = 8 rats per group.

Figure 3

Axonal and SYN labeling in the peri-infarcted area. (a) Representative images showing the BDA-labeled axons in the peri-infarcted area in the vehicle and TXL groups. Scale bar = 100 μm. (b) Compared with the vehicle groups (0.0302 ± 0.0037), TXL significantly increased the density of BDA-labeled fibers (0.0591 ± 0.0055) in the peri-infarcted area at 28 days after dMCAO (*P < 0.05). (c) Representative images of SYN and DAPI merge in the peri-infarcted area in vehicle and TXL groups. Scale bar = 40 μm. (d) Compared with the vehicle groups (0.05 ± 0.0165), TXL significantly increased SYN expression (0.0714 ± 0.0264) in the peri-infarcted area at 28 days after dMCAO (*P < 0.05). n = 10 samples per group. Red mass = infarcted area; Green arrow = BDA injection site; yellow square = sample site.

Figure 4

Axonal labeling in the corpus callosum. (a) Representative images showing the transcallosal axons in the middle portion of corpus callosum in the vehicle and TXL groups. (b) There are no significant differences with respect to axonal quantification in the corpus callosum between rats treated with vehicle (0.1983 ± 0.0222) or TXL (0.2121 ± 0.0243) (*P > 0.05). Scale bar = 50 μm. n = 10 samples per group. Red mass = infarcted area; green arrow = BDA injection site; yellow square = sample site.

Figure 5

Axonal labeling of the CST at the cervical level. (a) Representative images showing the axons of the CST at the cervical level in vehicle and TXL groups. (b) Schematic diagram showing the sample site of the CST. CC: corpus callosum. Scale bar = 150 µm. (c) There are no significant differences with respect to CST axonal density quantification between rats treated with vehicle (1945.63 ± 374.85) or TXL (2084.25 ± 377.55) (*P > 0.05). Scale bar = 50 μm. n = 8 rats per group. Data expressed as mean ± standard deviation. *P < 0.05 on ANOVA Tukey post hoc test. ^# P < 0.05 on ANOVA Tukey post hoc test.

Figure 6

Sprouting axons in the denervated spinal cord at the cervical level. (a) Schematic diagrams showing the axons sprouting across midline at the cervical level. (b) Representative images showing the axons in the denervated spinal cord at the cervical level in vehicle and TXL groups. (c) TXL significantly increased the number of lengthy axons within the gray matter in the denervated spinal cord at the cervical level after dMCAO (*P < 0.05). Scale bar = 50 μm. n = 8 rats per group. Vehicle groups: quotient of BDA-positive fibers’ number in A area (0.0176 ± 0.0025), B area (0.0265 ± 0.0038), and C area (0.00446 ± 0.0009). TXL groups: quotient of BDA-positive fibers’ number in A area (0.0305 ± 0.0073), B area (0.0506 ± 0.0098), and C area (0.0096 ± 0.0023).

Figure 7

Spouting axons and synapse formation in the denervated spinal cord at the cervical level. (a) Representative image showing SYN proteins yielded punctated signals, which scattered along BDA-labeled dendrites of spinal motoneurons in the vehicle rats. (b) Representative image showing substantial SYN closely encircling the dendrites in the denervated spinal cord of TXL-treated rats. Scale bar = 10 μm. Yellow square = sample site.