Protective efficacy of a single salvianolic acid A treatment on photothrombosis-induced sustained spatial memory impairments

Abstract

With respect to the high burden of ischemic stroke and the absence of pharmacological treatment for promoting rehabilitation, promising candidates with specific effects on long-term functional recovery are highly desired. Candidates need reasonable experimental paradigms to evaluate the long-term functional outcome focused on ischemia-induced sensorimotor and memory deficits. "Danshen", a traditional Chinese herb, has long been used to treat coronary and cerebral vascular diseases as well as dementia. Salvianolic acid A (SAA), one of the major active ingredients of Danshen, was demonstrated to be effective in protecting against cerebral ischemic injury. Here, employing an experimental stroke model induced by photothrombosis in the unilateral frontal cortex of rats, we investigated whether SAA has long-term protective effects on ischemia-induced sensorimotor and memory deficits in our behavioral tests. The results indicated that a single SAA treatment improved the cortical ischemia-induced sensorimotor deficits during 15 days' cylinder test period, and alleviated ischemia-induced sustained spatial memory impairments during the 2 months' dependent Morris Water Maze (MWM) tests. In addition, either ischemic injury or SAA treatment did not show any changes compared with sham group in other behavioral tests including rotarod tests, swimming speed in MWM tests, open field tests, elevated plus maze tests, treadmill tests and forced swimming tests. The results reveal that the cognitive deficits are not the results of animal's anxiety or confounding motor impairments. Overall, the present paradigm appears suitable for the preclinical evaluation of the long-term effects of pharmacological treatments on ischemic stroke. Meanwhile, SAA might have therapeutic potential for the treatment of memory deficits associated with ischemic stroke.

Keywords: dementia; ischemic stroke; memory impairments; salvianolic acid A.

Figure 1

Macroscopic appearance of cortical photothrombotic injury and the effect of SAA on the injury volume. Notes: (A) Schematic diagram of the injury locations on the dorsal surfaces and coronal sections of the rat brain; (B) The representative lesion pictures of ischemia treated with Saline and SAA. PFA-perfused brains and coronal section stained with Nissl staining showing a typical infarct in the frontal cortex. The borders of the infarct are marked with dotted lines. Note that all infarcts affected the frontal cortex (Fr1 and Fr2), and part of the forelimb area of the parietal cortex (FL). a, Fr1; b, Fr2; c, FL. (C) Molecular structure of SAA. (D) The effect of SAA on injury volume (mm³). Isch + Saline, ischemia control, n=9; Isch + SAA, ischemia treated with SAA, n=9. Data are given as mean ± SEM. *P<0.05 (Student’s t-test). Abbreviations: PFA, paraformaldehyde; SAA, Salvianolic acid A; SEM, standard error of the mean.

Figure 2

SAA treatment alleviates sensorimotor deficits following cortical photothrombosis. Notes: (A) The procedure for the sensorimotor test (sham, n=9; Isch + Saline, ischemia control, n=9; Isch + SAA, ischemia treated with SAA, n=9). (B and C) Compared with sham, rats with photothrombotic injury exhibited mild sensorimotor deficits in the cylinder test. (B) Forelimb use asymmetry score. (C) Sliding movements of contralateral forelimb. (D) Rotarod test. Data are given as mean ± SEM. ^###P<0.001 (repeated-measures ANOVA); *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA). Abbreviations: ANOVA, analysis of variance; SAA, Salvianolic acid A; SEM, standard error of the mean.

Figure 3

SAA treatment reversed spatial memory impairment in the MWM following cortical photothrombosis. Notes: (A) The procedure for behavioral study (sham, n=9; Isch + Saline, ischemia control, n=9; Isch + SAA, ischemia treated with SAA, n=9). (B and C) The learning curves in MWM. (B) Latency to find the platform. (C) Velocity. (D–F) Probe test with the platform removed. (D) Latency to reach the platform area, (E) Amount of platform crossings. (F) Time spent in the target quadrant. (G) Visible platform training. Data are given as mean ± SEM. ^#P<0.05, ^##P<0.01, ^###P<0.001 (repeated-measures ANOVA); *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA or κ Independent Sample test). Abbreviations: ANOVA, analysis of variance; MWM, Morris Water Maze; SAA, salvianolic acid A; SEM, standard error of the mean.

Figure 4

Effects of cortical photothrombotic injury and SAA treatment on anxiety- and depression-like behavior. Notes: Injured rats treated with saline or SAA had normal endurance capacity in the treadmill test (A) and normal locomotor activity in the open field test (B) as measured by total distance moved in 30 min. Injured rats treated with saline or SAA displayed normal levels of anxiety-like behaviors compared with sham rats as measured by the number of central zone entries in the open field test (C) and the elevated plus-maze test (D). Injured rats treated with saline or SAA exhibited normal levels of depression-like behaviors compared with sham rats as measured by the forced swimming test (E) and the sucrose preference test (F). Sham, n=9; Isch + Saline, ischemia control, n=9; Isch + SAA, ischemia treated with SAA, n=9. Data are given as mean ± SEM. Abbreviations: ANOVA, analysis of variance; SAA, Salvianolic acid A; SEM, standard error of the mean.