Astragaloside IV promotes cerebral tissue restoration through activating AMPK- mediated microglia polarization in ischemic stroke rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragaloside IV (AS), a key active ingredient obtained from Chinese herb Astragalus mongholicus Bunge, exerts potent neuroprotective and anti-inflammatory effects for treating neurodegenerative diseases. However, mechanisms of AS on improvement of ischemic brain tissue repair remain unclear. AIM OF THE STUDY: This research aims at using magnetic resonance imaging (MRI) to noninvasively determine whether AS facilitates brain tissue repair, and investigating whether AS exerts brain remodeling through adenosine monophosphate-activated protein kinase (AMPK) metabolic signaling regulating key glycolytic enzymes and energy transporters, thereby impacting microglia polarization. MATERIALS AND METHODS: Ischemic stroke model in male Sprague-Dawley rats were induced through permanent occlusion of the middle cerebral artery (MCAO). Infarct volume, the alterations of brain microstructure and nerve fibers reorganization were examined by multi-parametric MRI. The pathological damages of myelinated axons and microglia polarization surrounding infarct tissue were detected using pathological techniques. Furthermore, M1/M2 microglia polarization associated protein, glycolytic rate-limiting enzymes, energy transporters and AMPK/mammalian target of rapamycin (mTOR)/hypoxia inducible factor-1α (HIF-1α) signal were examined both in ischemic stroke rats and BV2 microglia treated with lipopolysaccharide (LPS) + interferon-γ (IFN-γ) by western blotting. RESULTS: MRI revealed that AS obviously decreased infarct volume, relieved brain microstructure damage and improved nerve fibers reorganization in ischemic stroke rats. Histological tests supported MRI findings. Notably, AS promoted microglia M2 and reduced M1 polarization, induced the AMPK activation accompanied with decreased levels of phosphorylated mTOR and HIF-1α. Moreover, AS suppressed the expression of glycolytic rate-limiting enzymes and energy transporters in ischemic stroke rats and BV2 microglia. In contrast, these beneficial effects were greatly blocked by AMPK inhibitor compound C. CONCLUSION: Overall, these results collectively suggested that AS facilitated tissue remodeling that may be partially through modulating polarization of microglia in AMPK- dependent metabolic pathways after ischemic stroke.

Berberine improves cognitive impairment by alleviating brain atrophy and promoting white matter reorganization in diabetic db/db mice: a magnetic resonance imaging-based study.

Diabetic cognitive impairment is a common complication in type 2 diabetes. Berberine (BBR) is an isoquinoline alkaloid that has been shown to have neuroprotective effects against diabetes. This study aimed to investigate the effect of BBR on the gray and white matter of the brain by using magnetic resonance imaging (MRI) and to explore the underlying mechanisms. The study used diabetic db/db mice and administered BBR (50 and 100 mg/kg) intragastrically for twelve weeks. Morris water maze was applied to examine cognitive function. T2-weighted imaging (T2WI) was performed to assess brain atrophy, and diffusion tensor imaging (DTI) combined with fiber tracking was conducted to monitor the structural integrity of the white matter, followed by histological immunostaining. Furthermore, the protein expressions of the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT)/ glycogen synthase kinase-3ß (GSK-3ß) were detected. The results revealed that BBR significantly improved the spatial learning and memory of the db/db mice. T2WI exhibited ameliorated brain atrophy in the BBR-treated db/db mice, as evidenced by reduced ventricular volume accompanied by increased hippocampal volumes. DTI combined with fiber tracking revealed that BBR increased FA, fiber density and length in the corpus callosum/external capsule of the db/db mice. These imaging findings were confirmed by histological immunostaining. Notably, BBR significantly enhanced the protein levels of phosphorylated AKT at Ser473 and GSK-3ß at Ser9. Collectively, this study demonstrated that BBR significantly improved the cognitive function of the diabetic db/db mice through ameliorating brain atrophy and promoting white matter reorganization via AKT/GSK-3ß pathway.

Buyang Huanwu Decoction promotes neurovascular remodeling by modulating astrocyte and microglia polarization in ischemic stroke rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Buyang Huanwu Decoction (BYHWD), one of the most commonly utilized traditional Chinese medicine prescription for treatment of cerebral ischemic stroke. However, the understanding of BYHWD on neurovascular repair following cerebral ischemia is so far limited. AIM OF THE STUDY: This research investigated the influence of BYHWD on neurovascular remodeling by magnetic resonance imaging (MRI) technology and revealed the potential neurovascular repair mechanism underlying post-treatment with BYHWD after ischemic stroke. MATERIALS AND METHODS: Male Sprague-Dawley rats were utilized as an ischemic stroke model by permanent occlusion of the middle cerebral artery (MCAO). BYHWD was intragastrically administrated once daily for 30 days straight. Multimodal MRI was performed to detect brain tissue injuries, axonal microstructural damages, cerebral blood flow and intracranial vessels on the 30th day after BYHWD treatment. Proangiogenic factors, axonal/synaptic plasticity-related factors, energy transporters and adenosine monophosphate-activated protein kinase (AMPK) signal pathway were evaluated using western blot. Double immunofluorescent staining and western blot were applied to evaluate astrocytes and microglia polarization. RESULTS: Administration of BYHWD significantly alleviated infarct volume and brain tissue injuries and ameliorated microstructural damages, accompanied with improved axonal/synaptic plasticity-related factors, axonal growth guidance factors and decreased axonal growth inhibitors. Meanwhile, BYHWD remarkably improved cerebral blood flow, cerebral vascular signal and promoted the expression of proangiogenic factors. Particularly, treatment with BYHWD obviously suppressed astrocytes A1 and microglia M1 polarization accompanied with promoted astrocyte A2 and microglia M2 polarization. Furthermore, BYHWD effectively improved energy transporters. Especially, BYHWD markedly increased expression of phosphorylated AMPK, cyclic AMP-response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) accompanied by inactivation of the NF-κB. CONCLUSION: Taken together, these findings identified that the beneficial roles of BYHWD on neurovascular remodeling were related to AMPK pathways -mediated energy transporters and NFκB/CREB pathways.

Magnetic resonance imaging detects cerebral gray and white matter injury correlated with cognitive impairments in diabetic db/db mice.

Type-2 diabetes not only causes gray matter injury but also induces widespread white matter damages, which may contribute the cognitive impairments. This study aimed to assess the structural alterations of the gray and white matter in 20-week-old diabetic db/db mice using magnetic resonance imaging including T2-weighted imaging (T2WI) and diffusion tensor imaging (DTI), and to correlate them with the cognitive performance detected by Morris water maze (MWM). The results revealed impaired spatial learning and memory in db/db mice. T2WI detected severe brain atrophy involving the hippocampus and cortex after diabetes. DTI showed reduced fractional anisotropy (FA) in the cortex, hippocampus, corpus callosum/external capsule, and increased radial diffusivity in the corpus callosum/external capsule of the db/db mice. The immunostaining confirmed the MRI findings showing decreased cell density in the cortex, hippocampus, and reduced integrated optical density of Luxol fast blue staining in the corpus callosum/external capsule. The correlational analysis revealed that the T2WI-derived tissue atrophy and DTI-derived FA in the relevant gray matter and white matter significantly correlated with the behavior performance in the MWM test. Collectively, the present in vivo MRI detected varying degrees of structural abnormalities in the gray and white matter of db/db mice, which might be favorable predictors of diabetic cognitive dysfunction. Our findings might provide new clues for identifying gray and white matter damages associated with cognitive decline, which is imperative for the evaluation of potential pharmacological therapies in preclinical phase.

Tetramethylpyrazine promotes stroke recovery by inducing the restoration of neurovascular unit and transformation of A1/A2 reactive astrocytes.

2,3,5,6-Tetramethylpyrazine (TMP) as an active ingredient extracted from a traditional Chinese herbal medicine Ligusticum chuanxiong Hort. has been proved to penetrate blood-brain barrier (BBB) and show neuroprotective effects on cerebral ischemia. However, whether TMP could regulate astrocytic reactivity to facilitate neurovascular restoration in the subacute ischemic stroke needs to be urgently verified. In this research, permanent occlusion of the middle cerebral artery (MCAO) model was conducted and TMP (10, 20, 40 mg/kg) was intraperitoneally administrated to rats once daily for 2 weeks. Neurological function was evaluated by motor deficit score (MDS). Magnetic resonance imaging (MRI) was implemented to analyze tissue injury and cerebral blood flow (CBF). Magnetic resonance angiography (MRA) was applied to exhibit vascular signals. Transmission electron microscopy (TEM) was performed to detect the neurovascular unit (NVU) ultrastructure. Haematoxylin and eosin (HE) staining was utilized to evaluate cerebral histopathological lesions. The neurogenesis, angiogenesis, A1/A2 reactivity, aquaporin 4 (AQP4) and connexin 43 (Cx43) of astrocytes were observed with immunofluorescent staining. Then FGF2/PI3K/AKT signals were measured by western blot. Findings revealed TMP ameliorated neurological functional recovery, preserved NVU integrity, and enhanced endogenous neurogenesis and angiogenesis of rats with subacute ischemia. Shifting A1 to A2 reactivity, suppressing excessive AQP4 and Cx43 expression of astrocytes, and activating FGF2/PI3K/AKT pathway might be potential mechanisms of promoting neurovascular restoration with TMP after ischemic stroke.

Magnetic Resonance Imaging Investigation of Neuroplasticity After Ischemic Stroke in Tetramethylpyrazine-Treated Rats.

Ischemic stroke elicits white matter injury typically signed by axonal disintegration and demyelination; thus, the development of white matter reorganization is needed. 2,3,5,6-Tetramethylpyrazine (TMP) is widely used to treat ischemic stroke. This study was aimed to investigate whether TMP could protect the white matter and promote axonal repair after cerebral ischemia. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO) and treated with TMP (10, 20, 40 mg/kg) intraperitoneally for 14 days. The motor function related to gait was evaluated by the gait analysis system. Multiparametric magnetic resonance imaging (MRI) was conducted to noninvasively identify gray-white matter structural integrity, axonal reorganization, and cerebral blood flow (CBF), followed by histological analysis. The expressions of axonal growth-associated protein 43 (GAP-43), synaptophysin (SYN), axonal growth-inhibitory signals, and guidance factors were measured by Western blot. Our results showed TMP reduced infarct volume, relieved gray-white matter damage, promoted axonal remodeling, and restored CBF along the peri-infarct cortex, external capsule, and internal capsule. These MRI findings were confirmed by histopathological data. Moreover, motor function, especially gait impairment, was improved by TMP treatment. Notably, TMP upregulated GAP-43 and SYN and enhanced axonal guidance cues such as Netrin-1/DCC and Slit-2/Robo-1 but downregulated intrinsic growth-inhibitory signals NogoA/NgR/RhoA/ROCK-2. Taken together, our data indicated that TMP facilitated poststroke axonal remodeling and motor functional recovery. Moreover, our findings suggested that TMP restored local CBF, augmented guidance cues, and restrained intrinsic growth-inhibitory signals, all of which might improve the intracerebral microenvironment of ischemic areas and then benefit white matter remodeling.

The Impact of Ischemic Stroke on Gray and White Matter Injury Correlated With Motor and Cognitive Impairments in Permanent MCAO Rats: A Multimodal MRI-Based Study.

Background: Identifying the alterations of the cerebral gray and white matter is an important prerequisite for developing potential pharmacological therapy for stroke. This study aimed to assess the changes of gray and white matter after permanent middle cerebral artery occlusion (pMCAO) in rats using magnetic resonance imaging (MRI), and to correlate them with the behavior performance. Methods: Rats were subjected to pMCAO or sham surgery and reared for 30 days. Motor and cognitive function of the rats were examined by gait and Morris water maze (MWM) tests, respectively. Multimodal MRI was conducted to examine the functional and structural changes of the gray and white matter followed with luxol fast blue (LFB) staining. Results: The gait and MWM tests revealed significant motor and cognitive dysfunction in pMCAO rats, respectively. Magnetic resonance angiography presented abnormal intracranial arteries in pMCAO rats with reduced signal intensity of the anterior cerebral artery, anterior communicating cerebral artery, internal carotid artery, and increased basilar artery vessel signal compared with sham rats. Arterial spin labeling confirmed the decreased cerebral blood flow in the infarcted sensorimotor cortex and striatum. Structural T2-weighted imaging and T2 mapping showed brain atrophy and elevation of T2 value in the gray (sensorimotor cortex, striatum) and white (external capsule, internal capsule) matter of pMCAO rats. The results from diffusion tensor imaging (DTI) corresponded well with LFB staining showing reduced relative FA accompanied with increased relative AD and RD in the gray and white matter of pMCAO rats compared with sham rats. Fiber tracking derived from DTI further observed significantly reduced fiber density and length in the corresponding brain regions of pMCAO rats compared with sham rats. Specially, the DTI parameters (especially FA) in the relevant gray matter and white matter significantly correlated with the behavior performance in the gait and MWM tests. Conclusion: Collectively, the gray and white matter damages could be non-invasively monitored in pMCAO rats by multimodal MRI. DTI-derived parameters, particularly the FA, might be a good imaging index to stage gray and white matter damages associated with post-stroke motor and cognitive impairments.

Effect of Neurorepair for Motor Functional Recovery Enhanced by Total Saponins From Trillium tschonoskii Maxim. Treatment in a Rat Model of Focal Ischemia.

Trillium tschonoskii Maxim. (TTM), is a perennial herb from Liliaceae, that has been widely used as a traditional Chinese medicine treating cephalgia and traumatic hemorrhage. The present work was designed to investigate whether the total saponins from Trillium tschonoskii Maxim. (TSTT) would promote brain remodeling and improve gait impairment in the chronic phase of ischemic stroke. A focal ischemic model of male Sprague-Dawley (SD) rats was established by permanent middle cerebral artery occlusion (MCAO). Six hours later, rats were intragastrically treated with TSTT (120, 60, and 30 mg/kg) and once daily up to day 30. The gait changes were assessed by the CatWalk-automated gait analysis system. The brain tissues injuries, cerebral perfusion and changes of axonal microstructures were detected by multimodal magnetic resonance imaging (MRI), followed by histological examinations. The axonal regeneration related signaling pathways including phosphatidylinositol 3-kinases (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3 (GSK-3)/collapsin response mediator protein-2 (CRMP-2) were measured by western blotting. TSTT treatment significantly improved gait impairment of rats. MRI analysis revealed that TSTT alleviated tissues injuries, significantly improved cerebral blood flow (CBF), enhanced microstructural integrity of axon and myelin sheath in the ipsilesional sensorimotor cortex and internal capsule. In parallel to MRI findings, TSTT preserved myelinated axons and promoted oligodendrogenesis. Specifically, TSTT interventions markedly up-regulated expression of phosphorylated GSK-3, accompanied by increased expression of phosphorylated PI3K, AKT, but reduced phosphorylated CRMP-2 expression. Taken together, our results suggested that TSTT facilitated brain remodeling. This correlated with improving CBF, encouraging reorganization of axonal microstructure, promoting oligodendrogenesis and activating PI3K/AKT/GSK-3/CRMP-2 signaling, thereby improving poststroke gait impairments.

Trillium tschonoskii rhizomes' saponins induces oligodendrogenesis and axonal reorganization for ischemic stroke recovery in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Trillium tschonoskii Maxim. is one of traditional Chinese medical herbs that has been utilized to treat brain damages and cephalalgia. The neuroprotective effect of total saponins from Trillium tschonoskii rhizome (TSTT) has been demonstrated efficacy in rats following ischemia. However, the axonal remodeling effect of TSTT and the detailed mechanisms after ischemic stroke have not been investigated. AIM OF THE STUDY: We aimed to estimate therapeutic role of TSTT in axonal remodeling using magnetic resonance imaging (MRI) technique, and explored possible mechanisms underlying this process followed by histological assays in ischemic rats. METHODS: Male Sprague-Dawley (SD) rats underwent permanently focal cerebral ischemia induced by occluding right permanent middle cerebral artery. TSTT was intragastrically administrated 6 h after surgery and once daily for consecutive 15 days. Neurological function was assessed by the motor deficit score and beam walking test. T2 relaxation mapping and diffusion tensor imaging (DTI) were applied for detecting cerebral tissues damages and microstructural integrity of axons. Luxol fast blue (LFB) and transmission electron microscope (TEM) were performed to evaluate histopathology in myelinated axons. Double immunofluorescent staining was conducted to assess oligodendrogenesis. Furthermore, the protein expressions regarding to axonal remodeling related signaling pathways were detected by Western blot assays. RESULTS: TSTT treatment (65, 33 mg/kg) markedly improved motor function after ischemic stroke. T2 mapping MRI demonstrated that TSTT decreased lesion volumes, and DTI further confirmed that TSTT preserved axonal microstructure of the sensorimotor cortex and internal capsule. Meanwhile, diffusion tensor tractography (DTT) showed that TSTT elevated correspondent density and length of fiber in the internal capsule. These MRI measurements were confirmed by histological examinations. Notably, TSTT significantly increased Ki67/NG2, Ki67/CNPase double-labeled cells along the boundary zone of ischemic cortex and striatum. Meanwhile, TSTT treatment up-regulated the phosphorylation level of Ser 9 in GSK-3ß, and down-regulated phosphorylated ß-catenin and CRMP-2 expression. CONCLUSION: Taken together, our findings indicated that TSTT (65, 33 mg/kg) enhanced post-stroke functional recovery, amplified endogenous oligodendrogenesis and promoted axonal regeneration. The beneficial role of TSTT might be correlated with GSK-3/ß-catenin/CRMP-2 modulating axonal reorganization after ischemic stroke.

The three-phase enriched environment paradigm promotes neurovascular restorative and prevents learning impairment after ischemic stroke in rats.

Enriched environment (EE) with a complex combination of sensorimotor, cognitive and social stimulations has been shown to enhance brain plasticity and improve recovery of functions in animal models of stroke. The present study extended these findings by assessing whether the three-phase EE intervention paradigm would improve neurovascular remodeling following ischemic stroke. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO). A three-phase EE intervention paradigm was designed in terms of the different periods of cerebral ischemia by periodically rearranging the EE cage. Morris water maze (MWM) tests were performed to evaluate the learning and memory function. Multimodal MRI was applied to examine alterations to brain structures, intracranial vessels, and cerebral perfusion on the 31st day after MCAO. The changes of capillaries ultrastructure were examined by transmission electron microscope. Double-immunofluorescent staining was used to evaluate neurogenesis and angiogenesis. The expression of angiogenesis-related factors and neurovascular remodeling related signaling pathways including Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3 (GSK-3)/ß-catenin and the axon guidance molecules were detected by Western blot analysis. MRI measurements revealed that EE treatment significantly increased survival volume of cortex and striatum, improved cerebral blood flow (CBF), amplified anterior azygos cerebral artery (azACA), ipsilateral internal carotid artery (ICA) and anterior communicating artery (AComA) vessel signal compared with standard housed rats (IS). Consistent with these findings, EE reduced ischemic BBB damage of capillary, enhanced endogenous angiogenesis and modified the expression of VEGF, Ang-1 or Ang-2 in ischemic rats. Additionally, this proangiogenic effect was consistent with the increased progenitor cell proliferation and neuronal differentiation in the peri-infarct cortex and striatum after EE intervention. Specifically, EE intervention paradigm markedly increased expression of phosphorylated PI3K, AKT and GSK-3, but reduced phosphorylated ß-catenin. Moreover, the axon guidance proteins expression level was significant higher in EE group. In parallel to these findings, EE significantly enhanced recovery of lost spatial learning memory function in MCAO rats without affecting infarct size. Together, MRI findings along with histological results strongly supported that the three-phase EE paradigm benefited neurovascular reorganization and thereby improved poststroke cognitive function. Moreover, our findings suggest that this type of EE paradigm induced neurogenesis and angiogenesis, at least in part, via regulating PI3K/AKT/GSK-3/ß-catenin signaling pathway and activation of the intrinsic axonal guidance molecules in animal models of ischemic stroke.

Enhanced white matter reorganization and activated brain glucose metabolism by enriched environment following ischemic stroke: Micro PET/CT and MRI study.

Enriched environment (EE) is an effective rehabilitative protocol designed to enhance sensorimotor, cognitive and social stimulation. Current understanding of neuronal remodeling after EE intervention mainly derived from conventional histological methods. The efficacy of EE treatment on post-stroke brain reorganization still needed to be elucidated in vivo. This study aimed to examine the effects of post-ischemic EE treatment on the brain remodeling using magnetic resonance imaging (MRI) and 18F-FDG positron emission tomography (PET). Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO) and housed in standard environment (SE) or EE for consecutive 30 days. Cognitive testing was performed using the Morris water maze. White matter structural modifications were detected by MRI combined with histological analysis. In addition, PET scanning with 18F-FDG as a molecular probe was conducted to detect brain energy metabolism. Our results showed that EE significantly mitigated MCAO-induced impairments in spatial learning and memory, attenuated brain atrophy, protected white matter integrity, and enhanced white matter reorganization coupled with promoting oligodendrogenesis. In parallel to these findings, PET-MRI fused images showed that EE remarkably elevated regional cerebral metabolic rates of glucose in the lesioned sensorimotor cortex, striatum and corpus callosum/external capsule. More importantly, a strong correlation was demonstrated between glucose utilization and diffusion tensor imaging indices in the corresponding brain regions. The data herein indicated that improved global metabolism of glucose was a critical step in the reorganization of the white matter following post-stroke EE intervention. Although EE did not produce beneficial effects on restoring brain infarct volume, the broad range of structural and functional benefits observed in the present study raised the interesting possibility that EE might be an effective rehabilitative strategy for ischemic stroke.

An MRI Study of Neurovascular Restorative After Combination Treatment With Xiaoshuan Enteric-Coated Capsule and Enriched Environment in Rats After Stroke.

Xiaoshuan enteric-coated capsule (XSEC) is a Chinese medicinal compound widely used for treatment of ischemic cerebrovascular diseases. Enriched environment (EE) is an effective rehabilitative protocol designed to enhance sensorimotor, cognitive and social stimulation. This study aimed to apply magnetic resonance imaging (MRI) to non-invasively assess whether EE could augment the therapeutic benefits of XSEC on post-ischemic neurovascular remodeling. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO) and treated with XSEC and EE alone or combination for 30 consecutive days. Beam walking test and Morris water maze (MWM) test were performed to evaluate motor and cognitive function, respectively. Multimodal MRI was applied to examine alterations to brain structures, intracranial vessels, and cerebral perfusion on the 31st day after MCAO. Double-immunofluorescent staining was used to evaluate neurogenesis and angiogenesis. Western blot and RT-PCR were used to detect the expressions of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and the axon guidance molecules. Combination therapy with XSEC and EE significantly reduced cystic volume compared with XSEC and EE monotherapies. In line with this, combination treated rats performed better in the beam walking test and exhibited improved spatial memory in the probe trial of the MWM. Moreover, XSEC and EE combination treatment improved cerebral blood flow (CBF), amplified angiogenesis and upregulated VEGF protein levels. This proangiogenic effect was consistent with the increased progenitor cell proliferation and neuronal differentiation in the peri-infarct cortex and striatum. Specifically, the combined therapy of XSEC and EE markedly increased the Netrin-1 and Robo-1 protein expression levels compared with vehicle group, while no difference was observed between XSEC or EE monotherapy and vehicle group. Together, these findings indicate that the combination of XSEC and EE benefits neurovascular reorganization. This correlates with restoration of CBF, promotion of neurogenesis and angiogenesis, and activation of the intrinsic axonal guidance molecules, thereby facilitating greater physical rehabilitation after ischemic stroke.

MRI Evaluation of Axonal Remodeling After Combination Treatment With Xiaoshuan Enteric-Coated Capsule and Enriched Environment in Rats After Ischemic Stroke.

Xiaoshuan enteric-coated capsule (XSEC) is a compound Chinese medicine widely used for the treatment of ischemic stroke. Enriched environment (EE) is a rehabilitative intervention designed to facilitate physical, cognitive, and social activity after brain injury. This study aimed to assess whether the XSEC and EE combination could provide synergistic efficacy in axonal remodeling compared to that with a single treatment after ischemic stroke using magnetic resonance imaging (MRI) followed by histological analysis. Rats were subjected to permanent middle cerebral artery occlusion and treated with XSEC and EE alone or in combination for 30 days. T2-weighted imaging and diffusion tensor imaging (DTI) were performed to examine the infarct volume and axonal remodeling, respectively. The co-localization of Ki67 with NG2 or CNPase was examined by immunofluorescence staining to assess oligodendrogenesis. The expressions of growth associated protein-43 (GAP-43) and growth inhibitors NogoA/Nogo receptor (NgR)/RhoA/Rho-associated kinase2 (ROCK2) were measured using western blot and qRT-PCR. The Morris water maze (MWM) was performed to evaluate the cognitive function. MRI and histological measurements indicated XSEC and EE individually benefited axonal reorganization after stroke. Notably, XSEC + EE decreased infarct volume compared with XSEC or EE monotherapy and increased ipsilateral residual volume compared with vehicle group. DTI showed XSEC + EE robustly increased fractional anisotropy while decreased axial diffusivity and radial diffusivity in the injured cortex, striatum, and external capsule. Meanwhile, diffusion tensor tractography revealed XSEC + EE elevated fiber density in the cortex and external capsule and increased fiber length in the striatum and external capsule compared with the monotherapies. These MRI measurements, confirmed by histology, showed that XSEC + EE promoted axonal restoration. Additionally, XSEC + EE amplified oligodendrogenesis, decreased the expressions of NogoA/NgR/RhoA/ROCK2, and increased the expression of GAP-43 in the peri-infarct tissues. In parallel to these findings, rats treated with XSEC + EE exhibited higher cognitive recovery than those treated with XSEC or EE monotherapy, as evidenced by MWM test. Taken together, our data implicated that XSEC + EE exerted synergistic effects on alleviating atrophy and encouraging axonal reorganization partially by promoting oligodendrogenesis and overcoming intrinsic growth-inhibitory signaling, thereby facilitating higher cognitive recovery.

Effects of Xiaoshuan Enteric-Coated Capsule on White and Gray Matter Injury Evaluated by Diffusion Tensor Imaging in Ischemic Stroke.

Xiaoshuan enteric-coated capsule (XSECC) is a drug approved by the Chinese State Food and Drug Administration for the treatment of stroke. This study was to investigate the effects of XSECC on white and gray matter injury in a rat model of ischemic stroke by diffusion tensor imaging (DTI) and histopathological analyses. The ischemia was induced by middle cerebral artery occlusion (MCAO). The cerebral blood flow measured by arterial spin labeling was improved by treatment with XSECC on the 3rd, 7th, 14th and 30th days after MCAO. Spatiotemporal white and gray matter changes in MCAO rats were examined with DTI-derived parameters (fractional anisotropy, FA; apparent diffusion coefficient, ADC; axial diffusivity, λ//; radial diffusivity, λ⊥). The increased FA was found in the XSECC treatment group in the corpus callosum, external capsule and internal capsule, linked with the decreased λ//, λ⊥ and ADC on the 3rd day and reduced ADC on the 30th day in the external capsule, suggesting XSECC reduced the axon and myelin damage in white matter after stroke. The relative FA in the striatum, cortex and thalamus in XSECC treatment group was significantly increased on the 3rd, 7th, 14th and 30th days accompanied by the increased λ// on the 3rd day and reduced relative ADC and λ⊥ on the 30th day, indicating that XSECC attenuated cell swelling and membrane damage in the early stage and tissue liquefaction necrosis in the late stage in gray matter after stroke. Additionally, XSECC-treated rats exhibited increased mean fiber length assessed by diffusion tensor tractography. Moreover, histopathological analyses provided evidence that XSECC relieved nerve cell and myelin damage in white and gray matter after stroke. Our research reveals that XSECC could alleviate white and gray matter injury, especially reducing nerve cell damage and promoting the repair of axon and myelin after ischemic stroke.

Investigation of Ginkgo biloba extract (EGb 761) promotes neurovascular restoration and axonal remodeling after embolic stroke in rat using magnetic resonance imaging and histopathological analysis.

EGb 761 is a standardized natural extract from Ginkgo biloba leaf that has shown neuroprotective effects after ischemic stroke. This study aimed to use magnetic resonance imaging (MRI) to noninvasively evaluate whether EGb 761 promotes neurovascular restoration and axonal remodeling in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to permanent right middle cerebral artery occlusion (MCAO) and treated with EGb 761 (60 mg/kg) or saline intragastrically once daily for 15 days starting 6 h after MCAO. Functional recovery was analyzed using beam walking test. Multi-parametric MRI was applied to examine the alterations of gray-white structures, intracranial vessels, cerebral perfusion and axonal integrity, and followed with histological studies. Furthermore, the protein expression of axonal remodeling related signaling pathways including protein kinase B (AKT)/ glycogen synthase kinase-3ß (GSK-3ß)/ collapsin response mediator protein 2 (CRMP2) and NogoA/NgR were detected by Western blotting analysis. Multi-parametric MRI demonstrated that EGb 761 significantly reduced infarct volume, alleviated gray and white matter damage, and enhanced collateral circulation, cerebral perfusion and axonal remodeling. Histological examinations supported the MRI results. EGb 761 treatment facilitated behavioral recovery and amplified endogenous neurogenesis. Notably, treatment with EGb 761 significantly increased the levels of p-AKT, p-GSK-3ß and decreased the expression of p-CRMP2. In addition, EGb 761 treatment up-regulated the expression of growth associated protein 43 (GAP-43) and suppressed the activation of axonal growth inhibitory molecules NogoA and NgR. These findings indicated that EGb 761 enhanced neurovascular restoration, amplified endogenous neurogenesis and promoted axonal regeneration, which in concert may contribute to gray-white matter reorganization and functional outcome after stroke.

Xiaoshuan enteric-coated capsule alleviates cognitive impairment by enhancing hippocampal glucose metabolism, hemodynamics and neuroplasticity of rat with chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH) is identified as a critical risk factor of dementia in patients with cerebrovascular disease. Xiaoshuan enteric-coated capsule (XSECC) is a compound Chinese medicine approved by Chinese State Food and Drug Administration for promoting brain remodeling and plasticity after stroke. The present study aimed to explore the potential of XSECC to improve cognitive function after CCH and further investigate the underlying mechanisms. CCH was induced by bilateral common carotid artery occlusion (BCCAO) in rats. XSECC (420 or 140 mg/kg) treatment remarkably reversed BCCAO-induced cognitive deficits. Notably, after XSECC treatment, magnetic resonance angiography combined with arterial spin labeling noninvasively demonstrated significantly improved hippocampal hemodynamics, and 18F-FDG PET/CT showed enhanced hippocampal glucose metabolism. In addition, XSECC treatment markedly alleviated neuropathologies and improved neuroplasticity in the hippocampus. More importantly, XSECC treatment facilitated axonal remodeling by regulating the phosphorylation of axonal growth related proteins including protein kinase B (AKT), glycogen synthase kinase-3ß (GSK-3ß) and collapsin response mediator protein-2 (CRMP2) in the hippocampus. Taken together, the present study demonstrated the beneficial role of XSECC in alleviating BCCAO-induced cognitive deficits by enhancing hippocampal glucose metabolism, hemodynamics and neuroplasticity, suggesting that XSECC could be a useful strategy in cerebral hypoperfusion state and dementia.

Effects of total saponins from Trillium tschonoskii rhizome on grey and white matter injury evaluated by quantitative multiparametric MRI in a rat model of ischemic stroke.

ETHNOPHARMACOLOGICAL RELEVANCE: Trillium tschonoskii rhizome (TTR), a medicinal herb, has been traditionally used to treat traumatic brain injury and headache in China. Although the potential neuroprotective efficacy of TTR has gained increasing interest, the pharmacological mechanism remains unclear. Steroid saponins are the main bioactive components of the herb. AIM OF THE STUDY: To investigate the protective and repair-promoting effects of the total saponins from TTR (TSTT) on grey and white matter damages in a rat model of middle cerebral artery occlusion (MCAO) using magnetic resonance imaging (MRI) assay. MATERIALS AND METHODS: Ischemic stroke was induced by MCAO. TSTT and Ginaton (positive control) were administered orally to rats 6h after stroke and daily thereafter. After 15 days of treatment, the survival rate of each group was calculated. We then conducted neurological deficit scores and beam walking test to access the neurological function after ischemic stroke. Subsequently, T2-weighted imaging (T2WI) and T2 relaxometry mapping were performed to measure infarct volume and grey and white matter integrity, respectively. Moreover, diffusion tensor imaging (DTI) was carried out to evaluate the grey and white matter microstructural damage. Additionally, arterial spin labelling (ASL) - cerebral blood flow (CBF) and magnetic resonance angiography (MRA) images provided dynamic information about vascular hemodynamic dysfunction after ischemic stroke. Finally, haematoxylin and eosin (HE) staining was carried out to evaluate the stroke-induced pathological changes in the brain. RESULTS: The survival rate and neurological behavioural outcomes (Bederson scores and beam walking tests) were markedly ameliorated by TSTT (65mg/kg) treatment within 15 days after ischemic stroke. Moreover, T2WI and T2 relaxometry mapping showed that TSTT (65mg/kg) significantly reduced infarct volume and attenuated grey and white matter injury, respectively, which was confirmed by histopathological evaluation of brain tissue. The results obtained from DTI showed that TSTT (65mg/kg) not only significantly alleviated axonal damage and demyelination, but also promoted axonal remodelling and re-myelination. In addition, TSTT treatment also enhanced vascular signal density and increased CBF in rats after MCAO. CONCLUSION: Our results suggested the potential protective and repair-promoting effects of TSTT on grey and white matter from damage induced by ischemia. This study provides a modern pharmacological basis for the application of TSTT in managing ischemic stroke.