SAE improves neurological function and reduces muscle spasticity in PSS rats
The therapeutic effects of SAE on behavioral dysfunction in rats with post-stroke spasticity were evaluated using the Zea Longa score, modified Neurological Severity Score, and modified Ashworth Scale on Days 1, 3, 5, and 7 after model validation (n = 12 per group). The Zea Longa score and mNSS were used to assess neurological deficits, whereas the MAS score was used to evaluate muscle tone/spasticity. After modeling, rats in the Blank and Sham groups showed steady weight gain, normal behavior, and preserved activity. In contrast, rats in the MCAO group developed weight loss, lethargy, reduced food intake, and limited movement of the left limb. After treatment, rats in both the SAE and Baclofen groups gradually regained body weight and showed improved behavioral status compared with the MCAO group (Figure 2).
For the Zea Longa score, two-way repeated-measures ANOVA showed significant effects of time and group × time interaction (p < 0.001). Post hoc analysis showed no significant difference between the Blank and Sham groups at any time point (p = 1.000). In contrast, the MCAO, SAE, and Baclofen groups had significantly higher Zea Longa scores than the Blank and Sham groups throughout the observation period (p < 0.001). Compared with the MCAO group, neither the SAE group nor the Baclofen group showed a significant difference at Days 1 or 3 (p > 0.05). However, both treatment groups showed significantly lower scores than the MCAO group at Days 5 and 7 (p < 0.001). No significant difference was observed between the SAE and Baclofen groups at any time point (p. > 0.05) (Figures 1B, 2A–B).
For the mNSS, two-way repeated-measures ANOVA showed significant effects of group, time, and group × time interaction (p < 0.001). Post hoc analysis showed no significant difference between the Blank and Sham groups at any time point (p > 0.05). In contrast, the MCAO, SAE, and Baclofen groups had significantly higher mNSS scores than the Blank and Sham groups at all time points after model validation (p < 0.001). Compared with the MCAO group, the SAE group showed significantly lower mNSS scores at Days 3, 5, and 7, whereas the Baclofen group showed significantly lower scores at Days 5 and 7. By Day 7, both treatment groups exhibited marked improvement compared with the MCAO group (p. < 0.001) (Figures 1C, 2C–D).
For the MAS score, repeated-measures analysis showed no significant change over time in the Blank or Sham groups, whereas significant time effects were observed in the MCAO, SAE, and Baclofen groups. Post hoc analysis showed no significant difference between the Blank and Sham groups at any time point (p > 0.05). In contrast, the MCAO, SAE, and Baclofen groups had significantly higher MAS scores than the Blank and Sham groups at all time points (p < 0.001). Compared with the MCAO group, the SAE group showed significantly lower MAS scores at Days 5 and 7, whereas no significant difference was observed between the Baclofen and MCAO groups at any time point (p > 0.05). No significant difference was detected between the SAE and Baclofen groups at any time point (p. > 0.05) (Figures 1D, 2E–F).
SAE reduces cerebral infarct volume in PSS rats
TTC staining was performed to evaluate cerebral infarction, and infarct severity was quantified as the ratio of infarct volume to total brain volume (n = 3 per group). The Blank and Sham groups showed uniform red staining without pale infarct regions (p > 0.05), whereas the MCAO, SAE, and Baclofen groups displayed different degrees of ischemic damage (Figure 3A). Infarct volume was significantly higher in the MCAO group than in the Blank and Sham groups (p < 0.05) (Figure 3B). After treatment, both SAE and Baclofen significantly reduced infarct volume compared with MCAO (p < 0.05). Notably, infarct volume in the SAE group was lower than that in the Baclofen group (p. < 0.05). These results indicate that SAE effectively reduces cerebral infarct burden and contributes to neurological recovery in rats with PSS.
SAE attenuates neuronal injury in PSS rats
HE staining was used to evaluate neuronal injury in the motor cortex (n = 3 per group). The Blank and Sham groups displayed near-normal cortical morphology (Figure 3C). Neurons were abundant and regularly arranged, with round nuclei and uniform cytoplasm. No obvious necrosis, degeneration, edema, or inflammatory infiltration was observed, and glial cells were evenly distributed without clear proliferation. In contrast, the MCAO group showed marked pathological changes. Some neurons were shrunken and deeply stained, with pyknotic morphology (black arrows), while others showed cytoplasmic pallor and edema (yellow arrows). Tissue architecture became loose. Compared with the MCAO group, both the SAE and Baclofen groups showed milder injury. Neuronal arrangement was more regular, nuclear and cytoplasmic morphology was improved, and only a small number of pyknotic neurons remained (black arrows).
Nissl staining was then performed to assess neuronal survival in the infarct cortex (n = 3 per group) (Figure 3D). In the Blank and Sham groups, neurons were intact and orderly, and Nissl bodies were evenly distributed without obvious loss. In the MCAO group, neurons were loosely arranged and showed swelling, nuclear shrinkage, membrane disruption, and blurred axonal boundaries. Nissl bodies were reduced, unevenly stained, and partially dissolved (black arrows), with evident microglial proliferation (yellow arrows). Compared with the MCAO group, the SAE group showed clearer neuronal structure, fewer axonal breaks, and more preserved Nissl bodies. The Baclofen group also improved significantly and showed slightly greater Nissl body preservation than the SAE group. Taken together, HE and Nissl staining indicate that SAE attenuates cortical neuronal injury and improves ischemia-related histopathological damage, consistent with a neuroprotective effect. Because neuronal preservation supports synaptic remodeling, these findings also provide a structural basis for subsequent changes in synaptic plasticity-related proteins and for SAE's antispastic effects.
SAE restores synaptic structural integrity in PSS rats
TEM was used to evaluate synaptic ultrastructure in rats with PSS (n = 3 per group). Synapse number was quantified at 5,000× magnification. For each group, three neuropil fields from three samples were randomly selected, and mean synapse counts were compared by one-way ANOVA. Synapse number did not differ between the Blank and Sham groups (p > 0.05) (Figures 4A–4B). In contrast, the MCAO group showed a marked reduction in synapse number compared with both control groups (p. < 0.001).
Compared with the MCAO group, synapse number was significantly higher in the SAE and Baclofen groups (p < 0.05), with no significant difference between the two treatment groups (p. > 0.05). Synaptic morphology was further examined at 8,000× magnification (Figure 4C). In the Blank and Sham groups, synaptic structures were clear and intact, with clustered synaptic vesicles, narrow synaptic clefts, and uniform postsynaptic density (PSD). The MCAO group showed pronounced ultrastructural damage, including disorganized synaptic architecture, partial structural dissolution, reduced presynaptic vesicle density, widened synaptic clefts, and thinned PSD. Compared with the MCAO group, both SAE and Baclofen improved synaptic ultrastructure, with more preserved architecture, less vesicle dispersion, and tighter synaptic clefts. These findings indicate that SAE helps restore synaptic integrity and increase synapse number in PSS rats. Structurally, these changes support more efficient synaptic transmission and provide a morphological basis for enhanced synaptic plasticity and reduced limb spasticity.
SAE regulates synaptic plasticity-related proteins and neurotrophic factors
Western blotting was performed to quantify GAP-43, SYN, PSD-95, and GDNF expression (n = 3 per group) (Figure 5A). Protein levels did not differ between the Blank and Sham groups (p > 0.05). Compared with these two control groups, the MCAO group showed significantly lower expression of SYN, PSD-95, GAP-43, and GDNF (p < 0.05). Both SAE and Baclofen treatment significantly increased the expression of these proteins relative to the MCAO group (p < 0.05), with no significant difference between the two treatment groups (p. > 0.05) (Figures 5B–5E). These results indicate that SAE enhances the expression of synaptic plasticity-related proteins in the post-ischemic cortex and upregulates GDNF, supporting its role in neuroprotection and functional recovery.
Relationship between the PI3K/Akt pathway and synaptic plasticity proteins
Immunofluorescence staining was used to examine SYN and p-Akt (n = 3 per group). SYN (red) was mainly localized to presynaptic regions, whereas p-Akt (green) was primarily distributed in the cytoplasm and cell membrane. In the healthy cortex, p-Akt signal was observed around SYN-positive structures, showing clear co-expression (Figure 6). After SAE treatment, the expression of SYN and p-Akt in the motor cortex was visibly increased. These findings suggest coordinated activation of PI3K/Akt signaling and synaptic plasticity-related protein expression.
SAE activates the PI3K/Akt pathway
To investigate the effect of SAE on the PI3K/AKT pathway after ischemic stroke, Western blotting was performed to assess total AKT (t-AKT), phosphorylated AKT (p-AKT), total PI3K (t-PI3K), and phosphorylated PI3K (p-PI3K) (Figure 7A). PI3K phosphorylation (p-PI3K/t-PI3K) was significantly higher in the SAE group than in the MCAO group (p < 0.05) (Figure 7B). Akt phosphorylation level (p-Akt/t-Akt) was significantly higher in both the SAE and Baclofen groups than in the MCAO group (Figure 7C). Although the Baclofen group showed slightly lower PI3K and Akt phosphorylation levels than the SAE group, the differences between the two treatment groups were not statistically significant (p. > 0.05). These findings suggest that SAE exerts neuroprotective effects in PSS rats, at least in part, by regulating the PI3K/Akt pathway.
DATA AVAILABILITY:
The datasets generated and/or analyzed during the current study (including raw data of behavioral scores, histopathological staining, Western blot band intensities, and transmission electron microscopy images) have been submitted as Supplementary Image materials and folders called Supplementary Raw data 1–3.

Figure 1: Pathological and behavioral changes 7 days after modeling. (A) Experimental grouping and intervention protocol. (B) Comparison of Zea Longa scores at different time points after successful model validation (n = 12). (C) Comparison of mNSS scores at different time points after successful model validation (n = 12). (D) Comparison of modified MAS scores at different time points after successful model validation (n = 12). Abbreviations; mNSS =modified neurological severity score; MAS = modified Ashworth score. The schematic illustration was manually created by the authors using Adobe Illustrator; no online source images or stock-library graphics were used. Please click here to view a larger version of this figure.

Figure 2: Behavioral assessment at 3 and 7 days after successful model validation. (A) Zea Longa score at Day 3 after successful model validation (n = 12). (B) Zea Longa score at Day 7 after successful model validation (n = 12). (C) mNSS at Day 3 after successful model validation (n = 12). (D) mNSS at Day 7 after successful model validation (n = 12). (E) MAS score at Day 3 after successful model validation (n = 12). (F) MAS score at Day 7 after successful model validation (n = 12). Behavioral data were analyzed using two-way repeated-measures ANOVA followed by Sidak’s multiple-comparisons test. Statistical significance is indicated by asterisks for the indicated pairwise comparisons (* p < 0.05, ** p < 0.01, *** p. < 0.001). Abbreviations; mNSS = modified neurological severity score; MAS = modified Ashworth score. Please click here to view a larger version of this figure.

Figure 3: Cerebral infarct volume and histopathological changes after intervention. (A) TTC staining showing cerebral infarct volumes in the Blank, Sham, MCAO, SAE, and Baclofen groups (n = 3). (B) Quantification of infarct volume (%) in the five groups. Statistical significance is indicated by asterisks for the indicated pairwise comparisons (* p < 0.05, ** p < 0.01, *** p. < 0.001). (C) Representative hematoxylin and eosin (HE) staining images of each group (n = 3; scale bar = 50 µm). (D) Representative Nissl staining images of each group (n = 3; scale bar = 50 µm). Please click here to view a larger version of this figure.

Figure 4: Synaptic ultrastructure in the peri-infarct cortex. (A) Representative TEM images at 5.0 k magnification (scale bar = 1 µm). (B) Quantification of synapse number among five groups (n = 3). Statistical significance is indicated by asterisks for the indicated pairwise comparisons (* p < 0.05, ** p < 0.01, *** p. < 0.001). (C) Representative TEM images at 8.0 k magnification (scale bar = 500 nm). Abbreviations; TEM = transmission electron microscopy. Please click here to view a larger version of this figure.

Figure 5: Expression of synaptic plasticity-related proteins and GDNF in the cerebral cortex. (A) Representative Western blot images of GAP-43, SYN, and PSD-95 (n = 3). (B) Quantification of SYN expression in the cerebral cortex. (C) Quantification of GAP-43 expression. (D) Quantification of PSD-95 expression. (E) Quantification of GDNF expression. Statistical significance is indicated by asterisks for the indicated pairwise comparisons (* p < 0.05, ** p < 0.01, *** p. < 0.001). The GAPDH loading control bands shown here are from identical raw Western blot samples used for the PI3K/Akt protein detection in Figure 7, hence the same GAPDH blot is shared between Figure 5 and Figure 7. Abbreviations; GDNF = glial cell line-derived neurotrophic factor; SYN = synaptophysin; PSD-95 = postsynaptic density protein 95; GAPDH = glyceraldehyde-3-phosphate dehydrogenase. Please click here to view a larger version of this figure.

Figure 6: Immunofluorescence staining of SYN and p-Akt in the motor cortex. Representative immunofluorescence images showing SYN and p-Akt expression in each group (n = 3), with SYN in red, p-Akt in green, and DAPI in blue (scale bar = 50 µm). Abbreviations; DAPI = 4',6-diamidino-2-phenylindole; Akt = protein kinase B; p-Akt = phosphorylated protein kinase B. Please click here to view a larger version of this figure.

Figure 7: PI3K/Akt pathway-related protein expression in the cerebral cortex. (A) Representative Western blot images of total Akt, phosphorylated Akt, total PI3K, and phosphorylated PI3K (n = 3). (B) Quantification of PI3K phosphorylation (p-PI3K/t-PI3K) in the cerebral cortex of PSS rats. (C) Quantification of Akt phosphorylation (p-Akt/t-Akt) in the cerebral cortex of PSS rats. Statistical significance is indicated by asterisks for the indicated pairwise comparisons (* p < 0.05, ** p < 0.01, *** p. < 0.001). Abbreviations; PI3K = phosphatidylinositol 3-kinase; PSS = post-stroke spasticity. Please click here to view a larger version of this figure.

Figure 8: Proposed signaling mechanism of SAE in the treatment of post-stroke spasticity. Schematic diagram of the signaling mechanism underlying SAE in PSS treatment. In rats with ischemic brain injury, SAE intervention is accompanied by elevated GDNF levels and PI3K/AKT pathway activation. These changes correlate with upregulated expression of synaptic plasticity-related proteins (SYN, PSD-95, GAP-43), improved synaptic plasticity and motor neuron status, and relieved muscle spasticity. The schematic illustration was manually created by the authors using Adobe Illustrator; no online source images or stock-library graphics were used. Please click here to view a larger version of this figure.
| Score | Behavioral criteria |
| 0 | No observable neurological deficit |
| 1 | Failure to extend the contralateral forepaw fully |
| 2 | Circling to the contralateral side |
| 3 | Falling/leaning to the contralateral side when walking |
| 4 | No spontaneous walking with depressed level of consciousness |
Table 1: Zea Longa scale. Neurological deficits and limb spasticity were assessed on Days 1, 3, 5, and 7 after model validation using the Zea Longa score.
| Grade | Description |
| 0 | No increase in muscle tone. |
| 1 | Slight increase in muscle tone, manifested by a catch and release or by minimal resistance at the end of the range of motion (ROM) |
| 1+ | Slight increase in muscle tone, manifested by a catch followed by minimal resistance throughout the remainder (less than half) of the ROM |
| 2 | More marked increase in muscle tone through most of the ROM, but affected part(s) easily moved |
| 3 | Considerable increase in muscle tone; passive movement difficult |
| 4 | Considerable increase in muscle tone; passive movement difficult |
Table 2: Modified Ashworth scale. Neurological deficits and limb spasticity were assessed on Days 1, 3, 5, and 7 after model validation using the modified Ashworth Scale
| Tests (Max Points) | Scoring Criteria | Points |
| Motor tests(0–6) | | |
| A. Raising rat by the tail | Flexion of the forelimb | 1 |
| Flexion of the hindlimb | 1 |
| head movement >10° to the vertical axis within 30 s | 1 |
| B. Placing the rat on the floor | normal walk | 0 |
| inability to walk straight | 1 |
| circling toward the paretic side | 2 |
| falling to the paretic side | 3 |
| Sensory tests (0-2) | inability to respond to tactile stimuli (placing test) | 1 |
| inability to respond to proprioceptive stimuli (proprioceptive test) | 1 |
| Beam balance tests (0–6) | balances with steady posture | 0 |
| grasps side of beam | 1 |
| hugs beam and one limb falls down | 2 |
| hugs beam and two limbs fall down, or spins on beam (>60 s) | 3 |
| attempts to balance but falls off within 40 s | 4 |
| attempts to balance but falls off within 40 s | 5 |
| falls off, no attempt to balance | 6 |
| Reflexes and abnormal movements (0–4) | absence of pinna reflex | 1 |
| absence of corneal reflex | 1 |
| absence of startle reflex | 1 |
| seizures, myoclonus, or myodystony | 1 |
| (Composite of motor, sensory, beam balance, and reflex tests; 0 = normal, 18 = maximal deficit; higher scores indicate more severe neurological deficit.) | | |
Table 3: Modified neurological severity score. Neurological deficits and limb spasticity were assessed on Days 1, 3, 5, and 7 after model validation using the modified neurological severity score.
Supplementary Image materials: Raw image materials.Please click here to download this file.
Supplementary Raw Data 1: Raw data for HE, immunofluorescence, Western blot, Nissl, graphs, and TTC.Please click here to download this file.
Supplementary Raw Data 2: TEM imaging raw files and TEM details.Please click here to download this file.
Supplementary Raw Data 3: TEM imaging raw files.Please click here to download this file.