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

Abstract

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.

Keywords

diffusion tensor imaging gray matter stroke white matter

Introduction

Stroke is the third most common cause of death in the world and one of the leading causes of long-term disability, creating an urgent need for novel therapies^1

–5. Currently, many neuroprotective drugs that showed promise in preclinical testing failed in clinical stroke treatment^6,7. One of the main reasons is that these drugs mainly target pathogenic mediators in the protection of gray matter, but not white matter⁸. White matter plays an important role in signal transduction and communication across brain regions^9
–11 and is an important cause of cognitive decline and sensorimotor deficits in stroke^12
–14. Therefore, strategies that battle both gray and white matter injury are urgently needed for stroke therapies.

Xiaoshuan enteric-coated capsule (XSECC), a well-known traditional Chinese herbal formula for the treatment of stroke, has been approved by the Chinese State Food and Drug Administration¹⁵. Previous studies have reported that XSECC has multi-target neurovascular protective effects on ischemic stroke¹⁵. However, the potentially protective effects of XSECC on white and gray matter injury for the treatment of ischemic stroke have not yet been investigated.

Currently, the altered microstructures of white and gray matter caused by stroke are mainly detected by histological measurements which do not allow dynamic assessment at different time points in vivo¹⁵. Diffusion tensor imaging (DTI) is widely applied in the evaluation of white and gray matter injury, involving neuronal necrosis, axonal disconnection and myelin degradation and considered as a sensitive tool to monitor disease progression dynamically in vivo^16

–22. Therefore, in the present experiment, the DTI technique and histopathological analyses were employed to investigate the possible efficacy of XSECC on white and gray matter remodeling in a rat model of ischemic stroke.

Materials and Methods

Experimental Animals

The male Sprague–Dawley rats, weighing 280 ± 20 g, were purchased from the Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) and housed three per cage under a controlled temperature (21 ± 2°C) and humidity (55 ± 5%) environment with a 12-h light/dark cycle. All experimental procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the local Ethical Committee at the Capital Medical University, China (Permit Number: No. 2013-X-16).

Preparation and Quantitative Analysis of XSECC

XSECC was supplied by Sanmenxia Sinoway Pharmaceutical Co. Ltd. (Henan, China). The formula consists of seven Chinese herbs, including Radix Astragali, Pheretima, Radix Paeoniae Rubra, Radix Angelica Sinensis, Rhizoma Ligustici Chuanxiong, Semen Persicae, and Flos Carthami. Herbal sources, preparation method and quantitative analysis of XSECC were stated in detail in our previous study¹⁵.

Animal Grouping, Stroke Model Induction and Drug Administration

A total of 30 rats were randomly divided into three groups (10 rats per group), including sham group, model group and XSECC treatment group. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (MCAO) as previously described²³. Briefly, the right common carotid, external carotid and internal carotid arteries were exposed. The external carotid and internal carotid arteries were temporarily clamped using microsurgical clips. A 4-0 monofilament nylon suture was inserted into the internal carotid artery and gently advanced from the lumen of the internal carotid artery until it blocked the origin of the middle cerebral artery. Sham-operated rats underwent the same surgery but no occlusion procedure. The rats in the XSECC group were treated with XSECC dissolved in sterile saline at a dose of 420 mg/kg/day for 30 days as previously described¹⁵. Rats in the sham and model groups were administered with normal saline (10 ml/kg/day).

Magnetic Resonance Imaging Protocols

The rats were subjected to magnetic resonance imaging examination using a 7.0 T magnet (Bruker, Pharma Scan, Germany) on the 3rd (n=4, 7, 7 in the sham, model and XSECC groups respectively), 7th (n=4, 6, 6 in the sham, model and XSECC groups respectively), 14th (n=4, 3, 5 in the sham, model and XSECC groups respectively) and 30th days (n=4, 2, 4 in the sham, model and XSECC groups respectively) after MCAO. Rats were anesthetized using facemask inhalation of 1.5–2% isoflurane before scanning.

The cerebral blood flow (CBF) was measured by arterial spin labeling with echo-planar imaging fluid-attenuated inversion recovery sequences (TR=18,000 ms; TE = 25 ms; field of view = 3.0 × 3.0 cm; matrix size = 128 × 128; number of excitations = 1). The CBF was measured in the cortex, striatum and thalamus on both ipsilateral and contralateral sides. Then, the relative CBF (rCBF) was derived from the ratio of ipsilateral CBF to contralateral CBF¹⁵.

DTI was acquired using an axial single-shot spin echo-planar sequence (TR = 6300 ms; TE = 25 ms; 30 diffusion directions; two b values = 0 and 1000 s/mm²). A fractional anisotropy (FA) map, apparent diffusion coefficient (ADC) map and three eigenvector (λ1, λ2, λ3) maps were reconstructed with ParaVision software, version 5.1 (Bruker, Pharmascan, Germany). FA, ADC, λ1(axial diffusivity, λ_//), λ2, and λ3 were analyzed in the regions of interest (ROIs) drawn on white matter (corpus callosum, external capsule and internal capsule) and gray matter (cortex, striatum and thalamus) at the level of bregma −1.4 mm. The radial diffusivity (λ_⊥) was calculated using the equation: λ_⊥ = (λ2+λ3)/2. The relative FA (rFA) or relative ADC (rADC) was calculated by comparing the parameters in the lesion ROIs with the mirrored ROIs of the contralateral side^24,25. Diffusion tensor tractography (DTT) was reconstructed with Diffusion Toolkit and TrackVis software, Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Boston, MA, USA. Relative mean tract length was derived from the ratio of ipsilateral mean tract length to contralateral mean tract length²⁶.

Hematoxylin-eosin and Luxol Fast Blue Staining

Hematoxylin-eosin (HE) staining was used to evaluate the histopathological changes. On the 30th day after MCAO, rats were transcardially perfused with saline solution, followed by 1% glutaraldehyde and 4% paraformaldehyde (PFA) in 0.1 mol/l phosphate buffer. Subsequently, the brains were removed and fixed in 4% phosphate-buffered PFA. Then brain tissues were embedded in paraffin and cut into coronal sections. The paraffin sections were baked at 60°C for 30 min, dewaxed by xylene, dehydrated with gradient alcohol, washed with running water for 1 min, and stained with HE. Images were visualized under an optical microscope at 400× magnification. A total of six nonoverlapping regions were sampled for the cortex, striatum and thalamus. Cell counts were expressed as the mean number of viable neurons/mm² as previously described²⁷.

Luxol fast blue (LFB) staining was performed to visualize white matter structures through the detection of myelin on the 30th day after MCAO. The paraffin sections were first immersed in xylene followed by anhydrous ethanol gradient dehydration. Thereafter, the sections were stained overnight in 0.1% LFB solution (Sigma-Aldrich, St. Louis, MO, USA) at 60°C. After washing with 95% ethyl alcohol and distilled water, the sections were differentiated in 70% ethyl alcohol for 30 s. Differentiation was terminated by washing in distilled water until the unmyelinated tissue appeared white. Images were visualized under an optical microscope at 400×magnification. The severity of the myelin lesions was graded by LFB staining images as normal (0), disarranged nerve fibers (1), the formation of marked vacuoles (2) and disappearance of myelinated fibers (3) in accordance with a previous report²⁸.

Statistics Analysis

All statistical analyses were carried out by using SPSS 17.0 software (SPSS, Chicago, IL, USA). Data are expressed as means ± standard deviation (SD). The one-way analysis of variance followed by a Fisher’s Least Significant Difference test was used for within-group comparisons. Pearson correlations were calculated to examine the linear association between DTI parameter FA and the number of surviving neurons or myelin damage grade. A significant difference was considered if the p-value was less than 0.05.

Results

XSECC Improved CBF in the Striatum, Cortex and Thalamus after Stroke

The rCBF in the cortex, striatum and thalamus in the model group was significantly lower than in the sham group on the 3rd, 7th, 14th and 30th days post stroke (P < 0.01, Fig. 1). The rCBF increased significantly in the cortex, striatum and thalamus in the XSECC treatment group over the course of 30 days after stroke (P < 0.01 or P < 0.05, Fig. 1).

Fig 1.

The effects of XSECC on rCBF in the cortex, striatum and thalamus on the 3rd, 7th, 14th and 30th days after stroke. n=4, 7, 7 in the sham, model and XSECC groups respectively on the 3rd day; n=4, 6, 6 in the sham, model and XSECC groups respectively on the 7th day; n=4, 3, 5 in the sham, model and XSECC groups respectively on the 14th day; n=4, 2, 4 in the sham, model and XSECC groups respectively on the 30th day. Values are means ± SD. Asterisks indicates significant differences. *P < 0.05, **P < 0.01 versus the model group.

XSECC Reduced the Injury of the Corpus Callosum, External Capsule and Internal Capsule in White Matter After Stroke

Serious damage occurred in the ischemic hemisphere in the model group evidenced by the abnormal signals on FA and ADC maps. The damage signals were weakened by XSECC treatment on the 3rd, 7th, 14th and 30th days after MCAO (Fig. 2).

Fig 2.

The representative images of FA and ADC of the rat brains at the level of bregma −1.4 mm on the 3rd, 7th, 14th and 30th days. Serious damage occurred in the ischemic hemisphere in the model group evidenced by the obvious abnormal signals pointed with dotted lines on FA and ADC maps. The damage signals were weakened by XSECC treatment on the 3rd, 7th, 14th and 30th days.

The measurements of FA, ADC, λ_// and λ_⊥ were performed on the corpus callosum, external capsule and internal capsule in white matter on the 3rd, 7th, 14th and 30th days after MCAO (Fig. 3A). The FA value in the corpus callosum in the ischemic hemisphere of the model group was significantly lower than that of the sham group on the 3rd day (P < 0.01, Fig. 3B), but there was no significant difference in FA on the 7th, 14th and 30th days between the sham and model groups (Fig. 3B). After treatment with XSECC, FA was remarkably increased compared with the model group on the 3rd day (P < 0.01, Fig. 3B).

Fig 3.

The effects of XSECC on white matter on the 3rd, 7th, 14th and 30th days after stroke. (A) The regions of interest drawn on white matter (corpus callosum, external capsule and internal capsule). (B, C, D) The statistical analysis of FA, ADC, λ_// and λ_⊥ in the corpus callosum, external capsule and internal capsule on the 3rd, 7th, 14th and 30th days after stroke. n=4, 7, 7 in the sham, model and XSECC groups respectively on the 3rd day; n=4, 6, 6 in the sham, model and XSECC groups respectively on the 7th day; n=4, 3, 5 in the sham, model and XSECC groups respectively on the 14th day; n=4, 2, 4 in the sham, model and XSECC groups respectively on the 30th day. Values are means ± SD. Asterisks indicates significant differences. *P < 0.05, **P < 0.01 versus the model group.

In the external capsule, the FA value in the model group was significantly decreased compared with the sham group on the 3rd, 7th, 14th and 30th days (P < 0.01, Fig. 3C), indicating that the external capsule was seriously damaged over the course of 30 days. The decreased FA was linked with increased ADC, λ_// and λ_⊥ on the 3rd day (P < 0.01, Fig. 3C) and increased ADC on the 30th day after stroke (P < 0.01, Fig. 3C). In contrast, the FA value in the XSECC group was significantly increased compared with the model group on the 3rd, 7th, 14th and 30th days (P < 0.05, Fig. 3C) accompanied by the decreased ADC, λ_//, and λ_⊥ on the 3rd day (P < 0.05, Fig. 3C) and the reduced ADC on the 30th day (P < 0.01, Fig. 3C).

In the internal capsule, the FA value in the model group was significantly decreased compared with the sham group on the 3rd, 7th, 14th and 30th days (P < 0.01 or P < 0.05, Fig. 3D) with decreased λ_// on the 3rd day (P < 0.01, Fig. 3D), reduced ADC on the 7th day (P < 0.05, Fig. 3D) and increased ADC on the 30th day (P < 0.05, Fig. 3D). XSECC-treated rats exhibited significantly higher FA value compared with the model group on the 3rd, 7th, and 14th days (P < 0.01 or P < 0.05) and a trend to increased λ_// on the 3rd day (P < 0.05, Fig. 3D).

XSECC Reduced the Injury of the Cortex, Striatum and Thalamus in Gray Matter After Stroke

The measurements of FA, ADC, λ_// and λ_⊥ were performed on the cortex, striatum and thalamus in gray matter on the 3rd, 7th, 14th and 30th days after stroke (Fig. 4A). The rFA in the cortex was significantly decreased in the model group on the 3rd, 7th, 14th and 30th days compared with the sham group (P < 0.01, Fig. 4B), linked with reduced λ_// on the 3rd and 7th day (P < 0.01 or P < 0.05, Fig. 4B) and increased rADC, λ_// and λ_⊥ on the 14th and 30th days (P < 0.01, Fig. 4B). XSECC-treated rats performed increased rFA on the 3rd, 7th, 14th and 30th days (P < 0.01 or P < 0.05), linked with increased λ_// on the 3rd day (P < 0.01, Fig. 4B) and reduced rADC, λ_// and λ_⊥ on the 30th day (P < 0.05, Fig. 4B).

Fig 4.

The effects of XSECC on gray matter on the 3rd, 7th, 14th and 30th days after stroke. (A) The regions of interest drawn on gray matter (cortex, striatum and thalamus). (B, C, D) The statistical analysis of FA, rADC, λ_// and λ_⊥ in the cortex, striatum, and thalamus on the 3rd, 7th, 14th and 30th days after stroke. n=4, 7, 7 in the sham, model and XSECC groups respectively on the 3rd day; n=4, 6, 6 in the sham, model and XSECC groups respectively on the 7th day; n=4, 3, 5 in the sham, model and XSECC groups respectively on the 14th day; n=4, 2, 4 in the sham, model and XSECC groups respectively on the 30th day. Values are means ± SD. Asterisks indicates significant differences. *P < 0.05, **P < 0.01 versus the model group.

In the striatum, the rFA value in the model group was significantly decreased compared with the sham group on the 3rd, 7th, 14th and 30th days (P < 0.01, Fig. 4C) with decreased rADC, λ_// and λ_⊥ on the 3rd day (P < 0.01, Fig. 4C) and increased rADC, λ_// and λ_⊥ on the 14th and 30th days (P < 0.01 or P < 0.05, Fig. 4C). XSECC-treated rats showed increased rFA on the 3rd, 7th, 14th and 30th days (P < 0.01 or P < 0.05, Fig. 4C), linked with increased rADC, λ_// and λ_⊥ on the 3rd day (P < 0.01, Fig. 4C) and decreased rADC, λ_// and λ_⊥ on the 14th and 30th days (P < 0.01 or P < 0.05, Fig. 4C).

In the thalamus, the rFA was significantly decreased in the model group on the 3rd, 7th, 14th and 30th days after stroke compared with the sham group (P < 0.01, Fig. 4D) with reduced λ_⊥ on the 7th day (P < 0.01, Fig. 4D) and increased rADC and λ_⊥ on the 30th day (P < 0.01 or P < 0.05, Fig. 4D). The rFA in the XSECC treatment group was decreased over the course of 30 days along with increased λ_⊥ on the 7th day (P < 0.01, Fig. 4D) and reduced rADC and λ_⊥ on the 30th day (P < 0.01 or P < 0.05, Fig. 4D).

XSECC Reduced the Injury of White Matter Fiber Bundles

Fiber tractography was reconstructed to trace the spatial distribution of white matter fiber bundles and observe the changes of the fiber bundles integrity on the 3rd, 7th, 14th and 30th days after stroke. The white matter fibers in the ischemic hemisphere were sparser than in the contralateral hemisphere with abnormal morphology, partial curl, and even missing in the rats of the model group on the 3rd, 7th, 14th and 30th days (Fig. 5A). XSECC could reduce nerve fiber injury and protect the integrity of white matter nerve fiber bundles over the course of 30 days after ischemic stroke (Fig. 5A). Statistical analysis results showed that the relative mean tract length in the model group was significantly decreased on the 3rd, 7th, 14th and 30th days after MCAO compared with the sham group (P < 0.01, Fig. 5B) and it was remarkably increased by treatment with XSECC when compared with the model group (P < 0.01 or P < 0.05, Fig. 5B).

Fig 5.

The effects of XSECC on nerve fiber injury on the 3rd, 7th, 14th and 30th days after stroke. (A) The representative images of fiber reconstruction of the rat brains at the level of bregma −1.4 mm on the 3rd, 7th, 14th and 30th days. White matter fibers in the ischemic hemisphere were sparser than that in the contralateral hemisphere with abnormal morphology, partial curl, and even were missing in the rats of the model group on the 3rd, 7th, 14th and 30th days. (B) The statistical analysis of mean tract length (% of contralateral hemisphere) on the 3rd, 7th, 14th and 30th days after stroke. n=4, 7, 7 in the sham, model and XSECC groups respectively on the 3rd day; n=4, 6, 6 in the sham, model and XSECC groups respectively on the 7th day; n=4, 3, 5 in the sham, model and XSECC groups respectively on the 14th day; n=4, 2, 4 in the sham, model and XSECC groups respectively on the 30th day. Values are means ± SD. Asterisks indicates significant differences. *P < 0.05, **P < 0.01 versus the model group.

XSECC Reduced Histopathological Damage in White Matter After Stroke

The histopathological changes in white matter (corpus callosum, external capsule and internal capsule) on the 30th day after stroke were evaluated by HE and LFB staining (Fig. 6A). The well-defined white matter structures, including corpus callosum, external capsule and internal capsule, were seen in the sham group on HE and LFB staining images. However, the loss of white matter integrity in the external capsule and internal capsule was observed in the model group on both HE and LFB staining images. HE staining showed that rats in the model group had vacuolation in the external capsule and internal capsule. LFB staining displayed severe myelin disruptions in the external capsule and internal capsule in the model group. By contrast, vacuolation and myelin damage were alleviated in external capsule and internal capsule by XSECC treatment. Statistical analysis showed that the myelin damage grade was significantly increased in the external capsule and internal capsule of the model group when compared with the sham group on the 30th day after stroke (P < 0.01, Fig. 6B). However, XSECC treatment markedly reduced the myelin damage grade compared with the model group (P < 0.05, Fig. 6B). The myelin damage grade in the corpus callosum showed no significant changes among the different groups on the 30th day after MCAO. Correlational analysis revealed the myelin damage grade in the corpus callosum, external capsule and internal capsule was negatively correlated with the DTI parameter FA (P < 0.01, Fig. 6C).

Fig 6.

The effects of XSECC on the histopathological changes in white matter (corpus callosum, external capsule and internal capsule) on the 30th day after stroke. (A) Representative HE and LFB staining images in the corpus callosum, external capsule and internal capsule from the sham, model and XSECC group rats. The well-defined white matter structures, including corpus callosum, external capsule and internal capsule, were seen in the sham group on HE and LFB staining images. However, the loss of white matter integrity in the external capsule and internal capsule were observed in the model group on both HE and LFB staining images. HE staining showed that rats in the model group had vacuolation indicated by arrows in the external capsule and internal capsule. LFB staining displayed severe myelin disruptions indicated by arrows in the external capsule and internal capsule in the model group. By contrast, vacuolation and myelin damage were alleviated in external capsule and internal capsule by XSECC treatment. Images were visualized under an optical microscope at 400× magnification. (B) The statistical analysis of the myelin damage grade on the 30th day after stroke. n=4, 2, 4 in the sham, model and XSECC groups respectively. Values are means ± SD. Asterisks indicates significant differences. *P < 0.05, **P < 0.01 versus the model group. (C) Pearson correlational analysis of the myelin damage grade and DTI parameter FA. A negative correlation was found between myelin damage grade and FA in the corpus callosum, external capsule and internal capsule.

XSECC Alleviated Histopathological Damage in Gray Matter After Stroke

The histopathological changes in gray matter (cortex, striatum and thalamus) on the 30th day after stroke were evaluated by HE staining (Fig. 7A). It showed that tissues and cells with normal histomorphology in the cortex, striatum and thalamus in the sham group. By contrast, the brain tissues in the model group exhibited obvious pathological abnormalities with loosely arranged neurons, pyknotic nucleus and loss or dark color staining in the cortex, striatum and thalamus (Fig. 7A). However, these histopathological alterations were reversed following treatment with XSECC (Fig. 7A). Quantitative analysis revealed that the number of surviving neurons in the cortex, striatum and thalamus was markedly reduced in the model rats on the 30th day after stroke, as compared with the sham rats (P < 0.01, Fig. 7B).

Fig 7.

The effects of XSECC on the histopathological changes in gray matter (cortex, striatum and thalamus) on the 30th day after stroke. (A) Representative HE staining images in the cortex, striatum and thalamus from the sham, model and XSECC group rats. The tissues and cells with normal histomorphology were observed in the cortex, striatum and thalamus in the sham group. By contrast, the brain tissues in the model group exhibited obvious pathological abnormalities with loosed arranged neurons, pyknotic nucleus and loss or dark color staining as indicated by arrows in the cortex, striatum and thalamus. However, these histopathological alterations were reversed following treatment with XSECC. Images were visualized under an optical microscope at 400× magnification. (B) The statistical analysis of the number of surviving neurons in the cortex, striatum and thalamus. n=4, 2, 4 in the sham, model and XSECC groups respectively. Values are means ± SD. Asterisks indicates significant differences. *P < 0.05, **P < 0.01 versus the model group. (C) Pearson correlational analysis of the number of surviving neurons and DTI parameter FA. A positive correlation was found between the number of surviving neurons and FA in the cortex, striatum and thalamus.

Treatment with XSECC significantly increased the number of surviving neurons in the cortex, striatum and thalamus compared with the model group on the 30th day post MCAO (P < 0.01 or P < 0.05, Fig. 7B), suggesting that XSECC protected neuronal cells of gray matter after stroke. Correlational analysis revealed the number of surviving neurons in the cortex, striatum and thalamus was positively correlated with the DTI parameter FA (P < 0.01 or P < 0.05, Fig. 7C).

Discussion

Ischemic stroke is accompanied by structural deformation and functional deficits²⁹. DTI enable in vivo assessment of the spatial and temporal pattern of structural and functional changes after stroke³⁰. DTI can quantitatively analyze the dispersion characteristics of water molecules in three-dimensional space¹⁶. The neuronal necrosis, gliosis, myelin sheath loss, axonal injury, nerve fiber loss, cellular inflammatory and other reactions after stroke can affect the diffusion rate of water molecules and thus DTI can accurately reflect the ultrastructure damage of white and gray matter^31,32. In the present study, DTI imaging was used to detect the FA, ADC, λ_// and λ_⊥ to evaluate the effects of XSECC on ultrastructural damage of white and gray matter in rats with ischemic stroke. First, our study demonstrated that DTI could monitor the evolution of white and gray matter reorganization dynamically and accurately after stroke. Then, DTI and histopathological analyses revealed that XSECC could alleviate white and gray matter injury evidenced by reducing nerve cell damage and promoting the repair of axon and myelin after ischemic stroke.

In white matter, FA, the ratio of the anisotropy of water molecules to the entire dispersion tensor³³, is closely related to the density and parallelism of fibers and the integrity of myelin, and can be used to reflect the integrity of white matter^22,34,35. Therefore, it is widely used in the evaluation of white matter damage in human and animal models^24,36,37. In this study, the decrease of FA caused by nerve fiber damage, axon degeneration and demyelination^26,38 in the corpus callosum only had obvious statistical significance on the 3rd day after stroke, while it had obvious statistical significance in the external capsule and internal capsule on the 3rd, 7th, 14th and 30th days after stroke. The results revealed that the corpus callosum was seriously damaged in the early stage while the external capsule and internal capsule were badly damaged at all stages of ischemic stroke. ADC is the diffusion rate of water molecules³⁹, and a reduced ADC value means increased limits in tissue. λ_// is the diffusion rate that parallels the axons and is mostly used to reflect the integrity of the axons⁴⁰. In early ischemia, λ_// will be significantly reduced due to severe cell edema and axonal damage⁴⁰; With ischemic time prolonging, λ_// will increase due to the gradually aggravated vasogenic edema and the loss of nerve fiber^40,41. λ_⊥ is the diffusion rate perpendicular to the axons and the change of λ_⊥ can reflect the pathological changes of demyelination^16,39,42. Generally, λ_⊥ will increase at the time of demyelination, myelination disorders and nerve fiber loss^39,43. In our study, FA was found to be decreasing accompanied by the increases of λ_//, λ_⊥ and ADC in the external capsule on the 3rd day after stroke, which was caused by the severe vasogenic edema associated with demyelination, impaired axonal injury and loss of fibers⁴⁴ that resulted in an increased diffusion rate of water molecules⁴⁴. FA decreased and ADC increased obviously in the external capsule on the 30th day after stroke, which was the result of the decrease of water molecule anisotropy and the increase of the diffusion rate, caused by the loss of fiber bundle and liquefaction and necrosis in a large area of the tissue⁴⁴. A decrease in FA value was linked with decreased λ_// in the internal capsule on the 3rd day after stroke, which may be due to the severe cell swelling that led to a narrow gap between the myelinated fiber bundles²⁶. After the administration of XSECC, the FA value in the corpus callosum, internal capsule and external capsule increased notably after stroke. The increased FA might arise from an increased number of aligned fibers or greater myelination^40,45. Additionally, the increased FA was linked with the decreased λ_//, λ_⊥ and ADC on the 3rd day and reduced ADC on the 30th day in the external capsule, indicating that drug treatment could reduce the damage of white matter axon and myelin, and maintain the integrity of the white matter structure and function.

DTT, also known as fiber tractography, is a noninvasive imaging method to show the travel and spatial distribution of brain white matter fiber bundles three-dimensionally in vivo, which can reflect the structure of the whole brain and the connectivity of different brain regions¹⁷. In this experiment, DTT showed that white matter fibers in the ischemic hemisphere were sparser than that of the contralateral hemisphere with abnormal morphology, partial curl, and were even missing in the rats of the model group on the 3rd, 7th, 14th and 30th days after stroke. The statistical results show that the mean tract length in the ischemic hemisphere in stroke group was significantly decreased and it was increased by treatment with XSECC on the 3rd, 7th, 14th and 30th days. The results showed that XSECC could reduce nerve fiber injury and protect the integrity of white matter nerve fiber bundles after ischemic stroke. Moreover, HE and LFB staining also provided evidence that XSECC relieved myelin damage in white matter respectively after stroke.

In gray matter, the rFA, rADC, λ_// and λ_⊥ decreased in the striatum and rFA and λ_// decreased in the cortex on the 3rd day after MCAO. The decrease in rFA was caused by the damaging of the cell membrane⁴⁶ and the decline in λ_// and λ_⊥ may be the result of severe cell swelling that limited the movement of water molecules³¹. On the 14th and 30th days post stroke, rFA in the striatum and cortex decreased significantly linked with increased rADC, λ_// and λ_⊥, which was caused by the necrotic liquefaction of infarcted tissue and increased permeability of the blood brain barrier with further development of vasogenic edema, allowing abundant proteins, electrolytes and water to enter the extracellular space and form cysts. This fluid-filled cystic area gave more space for the water free movement, resulting in a decrease in FA, and a significant increase in ADC, λ_// and λ_⊥ ⁴⁷. The ischemic injury was evident in the ipsilateral striatum and cortex following MCAO, while a secondary phase of injury also occurred in the ipsilateral thalamus remote from the primary injury in our study. The rFA in the ischemic far-thalamus was significantly lower on the 3rd, 7th, 14th and 30th days after stroke. The decreased rFA was accompanied by the increased rADC on the 14th and 30th days, which showed that obvious damage occurred in the distanced areas in the late stage of stroke, such as apoptosis, shrinkage and tissue liquefaction necrosis. Following treatment with XSECC, the rFA in the striatum, cortex and thalamus was significantly increased on 3rd, 7th, 14th and 30th days accompanied by the increased λ_// on the 3rd day in the striatum and cortex, and reduced rADC and λ_⊥ on the 30th day in the striatum, cortex and thalamus. The increased FA accompanied by other DTI parametric changes in gray matter potentially indicated that XSECC attenuated cell swelling and cell membrane damage in the early stage and mitigated tissue liquefaction necrosis in the late stage in gray matter after stroke.

Conclusions

The results of this study confirmed that DTI can accurately reflect the ultrastructure damage to white and gray matter after stroke. More importantly, we found that XSECC could alleviate the ultrastructural damage of white and gray matter after ischemic stroke, especially by reducing nerve cell damage and promoting the repair of axon and myelin injury, so as to promote the recovery of brain function.

Footnotes

Author Contributions

Jian Zhang, Shengpan Chen and Weilong Shi contributed equally to this work.

Ethical Approval

All procedures on animals in this study were in compliance with the ethical guidelines for researchers from the International Council for Laboratory Animal Science (ICLAS) and were approved by the Ethics Committee of Capital Medical University (No. 2013-X-16).

Statement of Human and Animal Rights

All experiments and animal rights were in compliance with relevant guidelines and regulations.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (Grant nos. 81473745 and 81774381) and the Beijing Municipal Natural Science Foundation (Grant no. 7172034).

ORCID iD

Hui Zhao

References

Zhang

Chopp

. Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic. Lancet Neurol. 2009;8(5):491–500.

Emmrich

Neher

Boehm-Sturm

Endres

Dirnagl

Harms

. Stage 1 registered report: effect of deficient phagocytosis on neuronal survival and neurological outcome after temporary middle cerebral artery occlusion (tMCAo). F1000Res. 2017;6:1827.

Jiang

Xia

Hong

Zhang

Gao

Chen

Shi

. Delayed docosahexaenoic acid treatment combined with dietary supplementation of omega-3 fatty acids promotes long-term neurovascular restoration after ischemic stroke. Transl Stroke Res. 2016;7(6):521–534.

Watson

Diamandis

Gonzales-Portillo

Reyes

Borlongan

. Melatonin as an Antioxidant for Stroke Neuroprotection. Cell Transplant. 2016;25(5):883–891.

Wei

Chen

Liu

Zhong

Zhang

Wang

Wei

. Enhanced Neurogenesis and collaterogenesis by sodium danshensu treatment after focal cerebral ischemia in mice. Cell Transplant. 2018;27(4):622–636.

McCracken

Fowler

Dewar

Morrison

McCulloch

. Grey matter and white matter ischemic damage is reduced by the competitive AMPA receptor antagonist, SPD 502. J Cereb Blood Flow Metab. 2002;22(9):1090–1097.

Gladstone

Black

Hakim

. Toward wisdom from failure: lessons from neuroprotective stroke trials and new therapeutic directions. Stroke. 2002;33(8):2123–2136.

Ouyang

Zhang

Cheng

BCY

Zhan

Yang

Zou

Zhao

. 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. J Ethnopharmacol. 2018;215:199–209.

Jiang

Wei

Hong

Zhang

Gao

Chen

Shi

. A Post-stroke therapeutic regimen with omega-3 polyunsaturated fatty acids that promotes white matter integrity and beneficial microglial responses after cerebral ischemia. Transl Stroke Res. 2016;7(6):548–561.

10.

Fields

. A new mechanism of nervous system plasticity: activity-dependent myelination. Nat Rev Neurosci. 2015;16(12):756–767.

11.

Wang

Shi

Leak

Liou

AKF

Badylak

Liu

Zhang

Chen

. Implantation of brain-derived extracellular matrix enhances neurological recovery after traumatic brain injury. Cell Transplant. 2017;26(7):1224–1234.

12.

Qiao

Meng

Scobie

Foniok

Tuor

. Magnetic resonance imaging of differential gray versus white matter injury following a mild or moderate hypoxic-ischemic insult in neonatal rats. Neurosci Lett. 2004;368(3):332–336.

13.

Zamboni

Griffanti

Jenkinson

Mazzucco

Kuker

Pendlebury

Rothwell

. White matter imaging correlates of early cognitive impairment detected by the montreal cognitive assessment after transient ischemic attack and minor stroke. Stroke. 2017;48(6):1539–1547.

14.

Ahn

Chen

Shin

Cho

Kim

Park

Lee

Tae

Lee

Byun

Jeong

Lee

Kim

Won

Choi

. Intravenously infused F3.Olig2 improves memory deficits via restoring myelination in the aged hippocampus following experimental ischemic stroke. Cell Transplant. 2016; 25(12):2129–2144.

15.

Zhang

Zou

Zhang

Wang

Lei

Wang

Ouyang

Zhang

Zhao

. Effects of Xiaoshuan enteric-coated capsule on neurovascular functions assessed by quantitative multiparametric MRI in a rat model of permanent cerebral ischemia. BMC Complement Altern Med. 2016;16:198.

16.

Wang

Tam

Lau

Cheung

Khong

. Characterization of white matter injury in a hypoxic-ischemic neonatal rat model by diffusion tensor MRI. Stroke. 2008;39(8):2348–2353.

17.

Nucifora

Verma

Lee

Melhem

. Diffusion-tensor MR imaging and tractography: exploring brain microstructure and connectivity. Radiology. 2007;245(2):367–384.

18.

Zhang

Aggarwal

Mori

. Structural insights into the rodent CNS via diffusion tensor imaging. Trends Neurosci. 2012;35(7):412–421.

19.

Granziera

D’Arceuil

Zai

Magistretti

Sorensen

de Crespigny

. Long-term monitoring of post-stroke plasticity after transient cerebral ischemia in mice using in vivo and ex vivo diffusion tensor MRI. Open Neuroimag J. 2007;1:10–7.

20.

Mori

Zhang

. Principles of diffusion tensor imaging and its applications to basic neuroscience research. Neuron. 2006;51(5):527–539.

21.

Bockhorst

Narayana

Liu

Ahobila-Vijjula

Ramu

Kamel

Wosik

Bockhorst

Hahn

Hasan

Perez-Polo

. Early postnatal development of rat brain: in vivo diffusion tensor imaging. J Neurosci Res. 2008;86(7):1520–1528.

22.

Ramu

Herrera

Grill

Bockhorst

Narayana

. Brain fiber tract plasticity in experimental spinal cord injury: diffusion tensor imaging. Exp Neurol. 2008;212(1):100–107.

23.

Longa

Weinstein

Carlson

Cummins

. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989;20(1):84–91.

24.

Watanabe

Honda

Fujii

Koyama

Matsuzawa

Tanaka

. Three-dimensional anisotropy contrast magnetic resonance axonography to predict the prognosis for motor function in patients suffering from stroke. J Neurosurg. 2001;94(6):955–960.

25.

Guo

Zheng

Duan

Chen

Gong

Tang

Wang

Cheng

. Diffusion tensor MRI for the assessment of cerebral ischemia/reperfusion injury in the penumbra of non-human primate stroke model. Neurol Res. 2011;33(1):108–112.

26.

Budde

Janes

Gold

Turtzo

Frank

. The contribution of gliosis to diffusion tensor anisotropy and tractography following traumatic brain injury: validation in the rat using Fourier analysis of stained tissue sections. Brain. 2011;134(Pt 8):2248–2260.

27.

Chen

Tai

Lee

Huang

Lee

Huang

. Cinnamophilin offers prolonged neuroprotection against gray and white matter damage and improves functional and electrophysiological outcomes after transient focal cerebral ischemia. Crit Care Med. 2011;39(5):1130–1137.

28.

Zhang

Zou

Ouyang

Zhan

Yang

Cheng

Wang

Zhang

Lei

Zhao

. 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. Biomed Pharmacother. 2018;103:989–1001.

29.

Cha

Kim

Jung

Han

Lee

. Altered white matter integrity and functional connectivity of hyperacute-stage cerebral ischemia in a rat model. Magn Reson Imaging. 2016;34(8):1189–1198.

30.

Dijkhuizen

van der Marel

Otte

Hoff

van der Zijden

van der Toorn

van Meer

. Functional MRI and diffusion tensor imaging of brain reorganization after experimental stroke. Transl Stroke Res. 2012;3(1):36–43.

31.

Tuor

Morgunov

Sule

Qiao

Clark

Rushforth

Foniok

Kirton

. Cellular correlates of longitudinal diffusion tensor imaging of axonal degeneration following hypoxic-ischemic cerebral infarction in neonatal rats. Neuroimage Clin. 2014;6:32–42.

32.

Zhang

Yao

Wang

Zhao

Shi

Zhao

Zhang

Qin

Cui

Liu

. Assessment of gray and white matter structural alterations in migraineurs without aura. J Headache Pain. 2017;18(1):74.

33.

Drobyshevsky

Derrick

Wyrwicz

Englof

Ullman

Zelaya

Northington

Tan

. White matter injury correlates with hypertonia in an animal model of cerebral palsy. J Cereb Blood Flow Metab. 2007;27(2):270–281.

34.

Ding

Jiang

Zhang

Ledbetter

Panda

Davarani

Athiraman

Ewing

Chopp

. Magnetic resonance imaging investigation of axonal remodeling and angiogenesis after embolic stroke in sildenafil-treated rats. J Cereb Blood Flow Metab. 2008;28(8):1440–1448.

35.

Jiang

Zhang

Ding

Silver

Zhang

Meng

Pourabdillah-Nejed

Wang

Savant-Bhonsale

Bagher-Ebadian

Arbab

Vanguri

Ewing

Ledbetter

Chopp

. MRI detects white matter reorganization after neural progenitor cell treatment of stroke. Neuroimage. 2006;32(3):1080–1089.

36.

Jiang

Zhang

Chopp

. MRI evaluation of white matter recovery after brain injury. Stroke. 2010;41(suppl 10):S112–S113.

37.

Ueda

Yamada

Kakuda

Abo

Senoo

. White matter structure and clinical characteristics of stroke patients: a diffusion tensor MRI study. Brain Res. 2016;1635:61–70.

38.

Cengiz

Uluc

Kendigelen

Akture

Hutchinson

Song

Zhang

Lee

Budoff

Meyerand

Sun

Ferrazzano

. Chronic neurological deficits in mice after perinatal hypoxia and ischemia correlate with hemispheric tissue loss and white matter injury detected by MRI. Dev Neurosci. 2011;33(3-4):270–279.

39.

Wang

Cai

Lau

Cheung

Khong

. Mild hypoxic-ischemic injury in the neonatal rat brain: longitudinal evaluation of white matter using diffusion tensor MR imaging. AJNR Am J Neuroradiol. 2009;30(10):1907–1913.

40.

Kujoth

Neves

Cikla

Akture

Uluc

Song

Hananya

Sadighi

Ferrazzano

Baskaya

. monitoring ischemic cerebral injury in spontaneously hypertensive rats by diffusion tensor imaging. Turk Neurosurg. 2016;26(4):500–512.

41.

Concha

Gross

Wheatley

Beaulieu

. Diffusion tensor imaging of time-dependent axonal and myelin degradation after corpus callosotomy in epilepsy patients. Neuroimage. 2006;32(3):1090–1099.

42.

Deo

Grill

Hasan

Narayana

. In vivo serial diffusion tensor imaging of experimental spinal cord injury. J Neurosci Res. 2006;83(5):801–810.

43.

Song

Sun

Ramsbottom

Chang

Russell

Cross

. Dysmyelination revealed through MRI as increased radial (but unchanged axial) diffusion of water. Neuroimage. 2002;17(3):1429–1436.

44.

Chan

Khong

Lau

Cheung

. Late measures of microstructural alterations in severe neonatal hypoxic-ischemic encephalopathy by MR diffusion tensor imaging. Int J Dev Neurosci. 2009;27(6):607–615.

45.

Engvig

Fjell

Westlye

Moberget

Sundseth

Larsen

Walhovd

. Memory training impacts short-term changes in aging white matter: a longitudinal diffusion tensor imaging study. Hum Brain Mapp. 2012;33(10):2390–406.

46.

Kim

Chang

Song

Choi

Son

Kim

Sohn

. Serial MR analysis of early permanent and transient ischemia in rats: diffusion tensor imaging and high b value diffusion weighted imaging. Korean J Radiol. 2013;14(2):307–315.

47.

Sizonenko

Camm

Garbow

Maier

Inder

Williams

Neil

Huppi

. Developmental changes and injury induced disruption of the radial organization of the cortex in the immature rat brain revealed by in vivo diffusion tensor MRI. Cereb Cortex. 2007;17(11):2609–2617.