†These authors contributed equally.
Background: Reperfusion therapy after ischemic cerebral stroke may
cause cerebral ischemia-reperfusion injury (CIRI), and cerebral edema is an
important factor that may aggravate CIRI. Our study aimed to dynamically monitor
the development of early cytotoxic edema after CIRI by magnetic resonance imaging
(MRI) and to validate it using multiple histological imaging methods.
Methods: Male Sprague Dawley rats were divided into sham and CIRI
groups. T2-weighted imaging (T2WI) and diffusion-weighted imaging (DWI)-MRI scans
were performed in the sham and CIRI groups after reperfusion. Relative apparent
diffusion coefficient (rADC) values were calculated and the midline shift (MLS)
was measured. A series of histological detection techniques were performed to
observe changes in the cerebral cortex and striatum of CIRI rats. Correlation
analysis of rADC values with aquaporin-4 (AQP4) and sodium-potassium-chloride
cotransport protein 1 (Na
With an increasingly aging population worldwide, the prevalence of ischemic stroke is also gradually increasing . While early intravenous thrombolysis or vascular intervention for embolization can save brain tissue on the verge of necrosis, it also can lead to cerebral ischemia-reperfusion injury (CIRI) . Cerebral edema is an important factor that may aggravate CIRI . Attention to brain edema progression and cytotoxic edema stages is important for improving patient prognosis [4, 5].
Magnetic resonance imaging (MRI) is a promising tool and a key component of numerous treatments and diagnostics, providing relatively stable and reproducible results . Studies have shown that T2-weighted imaging (T2WI) and diffusion-weighted imaging (DWI)-MRI can be used as reliable diagnostic methods to clinically determine the status of cerebral edema in patients with CIRI, providing reference information for determining subsequent treatment options . However, dynamic monitoring of CIRI patients has some limitations in clinical practice.
Astrocytes are the most abundant cell type in the mammalian brain, and astrocyte
swelling is the most important component of the early cytotoxic brain edema state
after CIRI . Cytoplasmic membranes of astrocytes are rich in water flux
channels such as aquaporin-4 (AQP4)  and sodium-potassium-chloride cotransport
protein 1 (Na
Clinical and translational studies have proved the ability of T2WI and DWI to monitor edema content and evolution . However, the dynamic evolution of cerebral edema, especially in the acute phase, is not fully understood. Therefore, in the present study, we applied T2WI and DWI-MRI methods to continuously and dynamically monitor the cerebral cortex and striatum of CIRI rats to quantitatively assess the alterations of cytotoxic edema, and compared in vivo images with histopathological results for a more in-depth analysis to understand the progression of cytotoxic edema after CIRI.
Specific-pathogen-free healthy adult male Sprague Dawley rats (8 weeks old;
250–280 g; Jinan Pengyue Laboratory Animal Breeding Co., Jinan, Shandong, China;
animal license: SCXK (Lu) 20220006) were housed under a 12 h light/dark cycle (22
Experiment design. Experimental timeline of the sham and CIRI groups, which underwent sham or MCAO operation, and a series tests of MR examination, TEM scans, HE staining, TUNEL staining, IF staining, and WB analysis were performed. CIRI, cerebral ischemia-reperfusion injury; IF, immunofluorescence; HE, hematoxylin-eosin; MCAO, middle cerebral artery occlusion; MR, magnetic resonance; TEM, transmission electron microscopy; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WB, western blot.
The neurological examination of CIRI rats was performed at CIRI-2 h according to the Longa scale . Neurological findings were graded on a five-point scale: 0, no neurological deficit; 1, mild focal neurologic deficit with the inability to extend the left forefoot; 2, moderate focal neurologic deficit with circling to the left; 3, severe focal deficit with falling to the left; and 4, inability to walk spontaneously and depressed level of consciousness. Scores of 1–3 were selected for inclusion in the study.
MRI examination was performed using a 7.0 T MRI scanner (BioSpec 70/20 USR; Bruker, Karlsruhe, Germany). Anesthesia was induced by a small animal anesthesia machine (R500, RWD, Shenzhen, Guangdong, China) using a mixture of 5% isoflurane and 95% oxygen, and maintained by a mixture of 2% isoflurane and 98% oxygen. The rats were placed in the center of the magnetic field. The regions of interest (ROIs) were outlined in the cerebral cortex and striatum. All ROI analyses were carried out in the ischemic right hemisphere and in the contralateral left hemisphere using the image analysis software ImageJ (ImageJ 1.6.0, National Institutes of Health, https://imagej.net/ij/index.html, Bethesda, MD, USA).
T2-weighted images were collected using a spin-echo sequence with the following
parameters: TR (repetition time) = 3000 ms, TE (echo time) = 34 ms, FOV (field of
view) = 3.3
The DWI parameters were: TR = 3000 ms, TE = 22 ms, FOV = 3.0
After MRI scans, all rats were injected with sodium pentobarbital (cat# P3761-5G; 58 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) and subsequently perfused with physiological saline and 4% paraformaldehyde (PFA) through the heart. The brain was removed and fixed in PFA for 24 h. Brain tissue was embedded in paraffin wax. The lateral ventricular level (2.2 mm anterior to 0.26 mm posterior to the bregmatic fontanelle) was localized and sliced using a paraffin slicer (Finesse 325; Thermo Scientific, Waltham, MA, USA) to make 4-µm-thick sections.
The paraffin sections were waxed, hydrated, and stained using a terminal deoxynucleotidyl transferase dUTP nick
end labeling (TUNEL) kit
(MK1014-100; Boster, Wuhan, Hubei, China), according to the manufacturer’s
instructions. The sections were then observed and photographed under an upright
optical microscope (BX51; Olympus, Tokyo, Japan). TUNEL
Paraffin sections of brain tissue were baked at a constant temperature of 60 ℃ for 1 h. The sections were then dewaxed using xylene, hydrated using gradient ethanol, and stained sequentially with hematoxylin-eosin (HE) (G1120; Solarbio, Beijing, China). The sections were then dehydrated in anhydrous ethanol and cleaned in xylene. Finally, the sections were sealed with neutral gum. The morphological changes of cells were observed under an upright optical microscope (BX51; Olympus, Tokyo, Japan).
After anesthesia, rats were perfused with 4% PFA and 2.5% glutaraldehyde
buffer (pH 7.4). Brain tissue specimens were collected, and cerebral cortex and
striatum on the ischemic side were separated and cut into small pieces (1
Immunofluorescence (IF) staining was performed on sections obtained from the sham and CIRI groups.
Brain tissue sections were dewaxed and hydrated sequentially, and antigen repair
was performed by the water bath thermal repair method. Rabbit anti-AQP4
(16473-1-AP, 1:1000; Proteintech, Wuhan, Hubei, China) and mouse anti-glial
fibrillary acidic protein (GFAP) (3670S, 1:300; Cell Signaling Technology,
Danvers, MA, USA) primary antibody mixture or rabbit anti-NKCC1 (13884-1-AP,
1:80; Proteintech) and mouse anti-GFAP primary antibody mixture were added as
needed, and refrigerated at 4 ℃ overnight. On the next day, goat anti-rabbit IgG
(ZF-0511, 1:200; Zhongshan Golden Bridge Biology Company, Beijing, China) and
goat anti-mouse IgG (ZF-0513, 1:200; Zhongshan Golden Bridge Biology Company)
fluorescent secondary antibody mixes were added, incubated for 1 h and protected
from light, and the slices were sealed with fluorescent blocker containing 4
Protein concentrations were quantified separately using a bicinchoninic acid
protein assay reagent kit (20201ES86, YEASEA, Shanghai, China). After
transferring the proteins to the polyvinylidene fluoride membrane, and the
membranes were blocked with 5% non-fat milk powder for 2 h. The strips were
incubated in rabbit anti-AQP4 (16473-1-AP, 1:5000; Proteintech, Wuhan, Hubei,
China) primary antibody, rabbit anti-NKCC1 (13884-1-AP, 1:1000; Proteintech,
Wuhan, Hubei, China) primary antibody, and mouse anti
Statistical analyses were performed using SPSS 22.0 software (SPSS Inc.,
Chicago, IL, USA). Shapiro Wilk analysis was used to verify the normality of the
measured data, and all normally distributed data are represented as mean
T2WI (Fig. 2A) and DWI-MRI (Fig. 2B,C) scans indicated that the brain tissue of
the sham group rats had uniform signal and bilateral symmetry, and no abnormal
signals were observed. At CIRI-2 h, abnormal signal areas of cerebral edema
appeared in the cerebral hemisphere on the ischemic side of the CIRI group, the
midline of the brain was shifted to the contralateral side, and the relative
apparent diffusion coefficient (rADC) values of the cerebral cortex and striatum
decreased compared with the sham group. The midline of the brain was further
shifted to the contralateral side at CIRI-6 h and 12 h, and the rADC values were
lower than before. At CIRI-24 h, extensive cerebral edema was seen in the
cerebral hemisphere on the ischemic side of the CIRI group, the MLS was
relatively large, and the rADC value reached a relatively low level. At CIRI-72
h, the MLS was reduced and the rADC value was increased compared to that at
CIRI-24 h (Fig. 2D–F, all *p
Dynamic multimodal MRI results. (A) T2WI images of the sham and
CIRI groups. (B) DWI images of the sham and CIRI groups. (C) ADC images of the
sham and CIRI groups. (D) Schematic diagram of the ROIs of the cortex and
striatum and computation of MLS. (E) Analysis of the relative MLS in the sham and
CIRI groups. (F) Analysis of the rADC in the sham and CIRI groups. Data are
presented as mean
HE staining showed that the nerve cells in the cortex and striatum of the sham group were regular in morphology and neatly arranged. At CIRI-24 h, the nerve cells had irregular morphology, disorganized arrangement, and loose structure, and some nuclei were deeply stained (Fig. 3A, n = 6).
The results of HE and TUNEL staining in the sham and CIRI-24 h
groups. (A) HE images of the sham and CIRI groups. (B) TUNEL images of the sham
and CIRI groups. (C) Analysis of the number of TUNEL
TUNEL assay showed that TUNEL
The perivascular astrocytic foot processes (APs) in the cerebral cortex and striatum were relatively thin and flat in the sham group, and the perivascular astrocytic APs were enlarged on the ischemic side of the CIRI-24 h group (Fig. 4, n = 6).
TEM images of the sham and CIRI-24 h groups. Enlarged areas
indicates an AP. Scale bar = 5 µm in 2000
IF double-label staining showed that the number of GFAP
IF staining of GFAP
According to western blot (WB) measurements, the expression of AQP4 and NKCC1 protein in the
ischemic cortex (Fig. 6A) and striatum (Fig. 6B) was significantly higher in the
CIRI-24 h groups than in the sham group (Fig. 6C,D, *p
WB analysis results of AQP4 and NKCC1 of the sham and CIRI-24 h
groups. (A) WB images of AQP4 and NKCC1 in the ischemic cortex of the sham and
CIRI-24 h groups. (B) WB images of AQP4 and NKCC1 in the ischemic striatum of the
sham and CIRI-24 h groups. (C) Comparison of AQP4 protein expression in the sham
and CIRI-24 h groups. (D) Comparison of NKCC1 protein expression in the sham and
CIRI-24 h groups. Data are presented as mean
Pearson’s correlation analysis and general linear regression showed that at each
time point after reperfusion, the number of GFAP
Correlation analysis of the number of GFAP
Ischemic stroke is an increasingly serious public health problem . Reperfusion therapy with thrombolysis or thrombus extraction has become the preferred method recommended by national guidelines , but the occurrence of cerebral edema after reperfusion can aggravate neurological impairment . There is therefore an urgent need to understand the dynamic progression of brain edema after CIRI to provide reliable information for clinical treatment .
In this study, we used MRI combined with multiple histological methods for the dynamic monitoring of brain edema after CIRI. We confirmed that the combined application of multi-scale and multi-parameter techniques can provide comprehensive information on CIRI rats. This is not only beneficial for exploring the pathophysiological changes in local lesions of brain diseases such as CIRI  but also helpful for improving our understanding and clinical prevention . Specifically, our MRI images showed significant enlargement of ischemic lesions in CIRI rats from the acute phase, which reached comparable severity at CIRI-24 h and then decreased. In addition, histologically, AQP4 and NKCC1 were closely associated with astrocytotoxic edema.
T2WI is commonly used to assess vasogenic edema and infarct area after CIRI , and the ADC value of DWI, a quantitative parameter, reflects the degree of diffusion restriction of water molecules due to cytotoxic edema . To summarize, we believe that MRI has high application value in CIRI, although histological methods are still needed to determine the accuracy and sensitivity of diagnoses . AQP4, which is mainly expressed in astrocytes, plays a key role in maintaining water balance, osmoregulation, energy metabolism, and cytotoxic edema after CIRI . NKCC1 is also an important transporter protein for astrocyte volume regulation, and its activation causes intracellular osmotic solute accumulation and co-transport of water molecules into the cell, leading to cytotoxic edema .
Our TEM results demonstrated that cytotoxic edema in the ischemic cortex and striatum occurred mainly in astrocytes, in accordance with a previous study . Other studies have found that AQP4  and NKCC1  both play crucial roles in early edema development following ischemia; however, this has not been the subject of a continuous dynamic observational study. The results of our study showed that the astrocyte water channel protein AQP4 and transporter protein NKCC1 expression increased in the ischemic side of the brain in CIRI rats and reached a relatively high level at 24 h in the cortex and at 12 h in the striatum. These results show that changes in the expression of brain edema-related proteins AQP4 and NKCC1 are closely related to astrocytic toxic edema. Moreover, correlation analyses showed that AQP4 and NKCC1 were negatively correlated with the quantitative parameter rADC values in the cortex and striatum. The above results verified the feasibility of using MRI combined with histologic multiscale imaging to monitor early cytotoxic edema after CIRI.
Our study also had some limitations. Cytotoxic edema was determined and evaluated by T2WI, DWI, and ADC-MRI combined with histological techniques from 2 h to 72 h after CIRI; however, further timepoints may need to be explored, and observations performed over a longer period of time.
Our results confirm that MRI combined with histological techniques can dynamically assess the progression of cytotoxic edema after CIRI in a clear and intuitive manner, and provide a scientific and theoretical basis for researchers and clinicians regarding the application of MRI techniques in this disease.
CIRI, cerebral ischemia-reperfusion injury; MRI, magnetic resonance imaging; T2WI, T2-weighted imaging; DWI, diffusion-weighted imaging; ADC, apparent diffusion coefficient; rADC, relative apparent diffusion coefficient; AQP4, aquaporin-4; NKCC1, sodium-potassium-chloride cotransport protein 1; GFAP, anti-glial fibrillary acidic protein; WB, western blot; HE, hematoxylin-eosin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; TEM, transmission electron microscopy; IF, immunofluorescence.
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.
XLW and LA conceived the design of the project. YHM and PLX contributed to writing and editing the manuscript. YHM performed the data analysis. YHM, CL, JFL, CML, and HMZ performed the experiments. PLX and HMZ contributed to the preparation of the MCAO animal model. XZW performed the magnetic resonance imaging. ZL and MY provided access to behavioral equipment and participated in behavioral testing. XLW supervised the project. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
The experimental procedures described in this study conformed to the requirements of the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were approved by the ethics committee of Weifang Medical University (approval No. 2020SDL052).
This work was supported by the National Natural Science Foundation of China (82071888), Natural Science Foundation of Shandong Province (ZR2020MH074; ZR2021MH351), Shandong Province Traditional Chinese Medicine Science and Technology Development Plan (2019-0417; Q-2023071), Shandong Province Science and Technology Development Plan (202009010562), Weifang City Science and Technology Development Plan (2021GX057, 2022YX042) and 2019 Young Creative Talent Induction Program for Higher Education Institutions.
The authors declare no conflict of interest. Lin An is currently the General Manager of Guangdong Weiren Meditech Co. Ltd, Foshan, Guangdong, China. He is a visiting professor hired by Weifang Medical University to provide us with technical support in this study. The animal experiments in our manuscript were conducted at Weifang Medical University and the instruments in the manuscript we used are all from Weifang Medical University. There is no conflict of interest with Guangdong Weiren Meditech Co. Ltd.
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