*Contributed equally
Although hypertonic saline (HS) has been extensively applied to treat brain edema in the clinic, the precise mechanism underlying its function remains poorly understood. Therefore, the aim of the present study was to investigate the therapeutic mechanism of HS in brain edema in terms of aquaporins and inflammatory factors. In the present study, traumatic brain injury (TBI) was established in male adult Sprague-Dawley rats, which were continuously administered 10% HS by intravenous injection for 2 days. In addition, brain edema and brain water content were detected by MRI and wet/dry ratio analysis and histological examination, respectively. Immunohistochemical staining for albumin and western blotting for occludin, zonula occludens-1 and claudin-5 was performed to evaluate the integrity of the blood-brain barrier. Aquaporin 4 (AQP4) expression was also analyzed using western blotting and reverse transcription-quantitative PCR, whilst interleukin (IL)-1β and NF-κB levels were measured using ELISA. It was demonstrated that HS treatment significantly reduced brain edema in TBI rats and downregulated AQP4 expression in cerebral cortical tissues around the contusion site. In addition, IL-1β and NF-κB levels were found to be downregulated after 10% HS treatment. Therefore, results from the present study suggested that HS may protect against brain edema induced by TBI by modulating the expression levels of AQP4, NF-κB and IL-1β.
Brain edema is a common complication following traumatic brain injury (TBI) around the world, which is associated with high disability, mortality and morbidity rates. In 2014, the Centers for Disease Control and Prevention documented 2.53 million TBI-related emergency department visits and there were approximately 288,000 TBI-related hospitalizations and 56,800 TBI-related deaths (
The AQP family consists of water channel proteins that participate in water homeostasis regulation (
A total of 60 adult male Sprague-Dawley (SD) rats (weight range, 220-270 g, 7 weeks old) were provided by Beijing Vital River Laboratory (Beijing, China). Animal treatments and experiments were carried out in accordance with the ‘Guide for the Care and Use of Laboratory Animals' of the National Institutes of Health (
A controlled cortical impact model of TBI was induced
Rats were randomized into three groups: i) Sham group (n=18); ii) vehicle-treated TBI group (n=21); and iii) HS-treated TBI group (n=21). For HS treatment, animals were given an intravenous injection of 10% HS (10 g NaCl in 100 ml deionized water) via the tail vein (0.08 ml/g; infusion rate, 0.3 ml/h) 3 h after the induction of TBI (
Sequential MRI was conducted in 21 rats (including 5, 8 and 8 from the sham, HS-treated and vehicle-treated groups, respectively) at 0 and 48 h after TBI using a 7.0 T scanner (BioSpec Products, Inc.) equipped with a four-channel phased array rat head coil.
T2-weighted turbo spin-echo images (T2WI) were obtained using the fast spin-echo sequence (under conditions of field-of-view, 35x35 mm; matrix, 256x256; number of scans, 20; slice thickness, 1 mm; echo time, 33 msec; repetition time, 2838.2 msec; number of averages, 1). The obtained images were analyzed using the OsiriX software (version 4.2.2;
Over the course of TBI, the contralateral brain area was determined according to the aforementioned method and was also delineated in the obtained images in accordance with rescaled drawings from the Paxinos and Watson Atlas (
where Vu and Ve represent the uncorrected and corrected lesion volumes, respectively and Vc and Vi represent the contralateral and ischemic hemisphere volumes, respectively.
The modified neurological severity score (mNSS) of each group was evaluated at 48 h following TBI in a blinded manner according to a previously described method (
Coronal sections of the cerebral cortical tissues around the contusion site were selected for H&E and IHC staining. Rats were sacrificed and perfused with PBS via the left ventricle followed by 4% paraformaldehyde for fixation. Then, the collected cerebral tissues were fixed at room temperature in buffered paraformaldehyde (4%) for 24 h embedded in paraffin and sectioned into 4-µm slices. H&E staining was performed after deparaffinization by xylene followed by a descending ethanol gradient at room temperature, where the stained tissues were examined under a light microscope (magnification, x400).
For IHC staining, rats were sacrificed and perfused with PBS via the left ventricle followed by 4% paraformaldehyde for fixation. Cortical tissues around the contusion site were collected and fixed at room temperature in 4% buffered paraformaldehyde for 24 h embedded in paraffin and sectioned into 4-µm slices, which were then deparaffinized using xylene and rehydrated by descending ethanol gradient. Hydrogen peroxide (3%) was diluted with deionized water and used to block endogenous peroxidase activity for 10 min at room temperature. Slices were incubated in citrate buffer (at pH=6.0) for 10 min at 98˚C for antigen retrieval and incubated with 10% goat serum (cat. no. ZLI-9022; Origene Technologies, Inc.) blocking solution for 20 min at 37˚C. Subsequently, the slices were incubated with the following primary antibodies: Rabbit anti-rat glial fibrillary acidic protein (GFAP; 1:2,000; cat. no. Z0334; Dako; Agilent Technologies, Inc.), chicken anti-rat albumin (1:1,000; cat. no. ab106582; Abcam) and rabbit anti-AQP4 (1:1,000; cat. no. ab128906; Abcam), overnight at 4˚C. The slices were then washed twice with PBS and then incubated at 37˚C with the relevant polymeric horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin G secondary antibody (Super Vision IHC kit; cat. no. SV0002; Boster Biological Technology) for 30 min at 37˚C. After staining with 3,3'-diaminobenzidine for ~3 min at room temperature, all slices were counterstained with hematoxylin for 2 min at 37˚C, dehydrated and mounted on coverslips for light microscopic examination. IHC staining for albumin, GFAP and AQP4 was examined by ImageJ software 1.8.0 (National Institutes of Health). In total, six visual fields from each slice (magnification, x400) were examined and the average optical density (OD) was determined for all images.
Brain edema was evaluated by calculating the BWC of all animals according to a previously described method (
Before the experiment ended, a 1-ml blood sample was collected from the rat tail vein under anesthesia as described previously (
The plasma level of IL-1β was determined using ELISA. Blood samples (1 ml) were collected from the rat abdominal aorta under anesthesia as aforementioned and then subjected to 15 min centrifugation at 800 x g at 4˚C. The plasma IL-1β concentration was measured using a commercial ELISA kit (cat. no. F15810, Shanghai Xitang Biological Technology Co., Ltd.) in accordance with the manufacturer's protocol. Then, the OD at 450 nm was determined using a microplate reader.
Total RNA was isolated from cells and brain tissue from the area surrounding the edema using RNAiso plus (cat. no. 9109; Takara Biotechnology Co., Ltd.). RNA quantification was carried out using a spectrophotometer (NanoDrop 2000; PEQLAB Biotechnologie GmbH), and cDNA was synthesized from 1.0 µg RNA by reverse transcriptase M-MLV (cat. no. 2641; Takara Biotechnology Co., Ltd.) according to manufacturer's protocol. The temperature and duration for reverse transcription was as follows: 42˚C for 10 min and 95˚C for 2 min. The primer sequences used for RT-PCR were as follows: NF-κB forward, 5'-ACAGCCTGGTAGTGCGGTCGT-3' and reverse, 5'-TCAGCAAGTGGCTAGTCTGT-3'; IL-1β forward, 5'-AAAAGCTTGGTGATGTCTGG-3' and reverse, 5'-TTT CAACACGCAGGACAGG-3'; AQP4 forward, 5'-CTTTCT GGAAGGCAGTCTCAG-3' and reverse, 5'-CCACACCGA GCAAAACAAAGAT-3'; and GADPH (the reference gene), forward, 5'-CGGATTTGGTCGTATTGGG-3' and reverse, 5'-CTGGAAGATGGTGATGGGATT-3'. qPCR was performed with SYBR® Premix Ex™ Taq (Takara Biotechnology Co., Ltd) using an ABI 7500 RT PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95˚C for 10 sec, followed by 40 cycles of 95˚C for 5 sec and 60˚C for 30 sec. A dissociation curve was used to examine the detected signal specificity, which constituted a single peak. The 2-ΔΔCq method was used for quantification of expression (
The perilesional brain tissue was isolated from each treatment group 2 days following TBI, which was subjected to homogenization with cytoplasmic buffer supplemented with KCl (10 mM), HEPES (10 mM, at pH=7.9), EGTA (0.1 mM), EDTA (0.1 mM), Nonidet P-40 (0.15%), DTT (1 mM), NaF (10 mM), β-glycerophosphate (50 mM), and Na3VO4 (5 mM) and phosphatase inhibitors (Roche Diagnostics). The homogenates were subjected to 30 min centrifugation at 12,000 x g at 4˚C, following which the supernatant was discarded and the pellet was not disturbed. To extract nuclear protein, a nuclear buffer was supplemented with NaCl (400 mM), HEPES (20 mM, at pH=7.9), EGTA (1 mM), EDTA (1 mM), Nonidet P-40 (0.50%), DTT (1 mM), NaF (10 mM), Na3VO4 (5 mM), β-glycerophosphate (50 mM), in addition to the protease inhibitor cocktail and the buffer was added to the aforementioned pellet. Subsequently, the homogenates were subjected to a further 15 min incubation on ice after vortexing at room temperature for 20 sec, which was repeated four times. The homogenates were then subjected to 15 min of centrifugation at 12,000 x g at 4˚C to assess the nuclear NF-κB.
AQP4 expression was measured using total protein extracts. Protein was collected from brain tissues around the injury site using a total protein extraction kit (Bei Jing Pu Li Lai Gene Technology Co., Ltd.) in accordance with the manufacturer's protocol. Then, the resultant homogenates were subjected to 30 min of centrifugation at 12,000 x g at 4˚C and the supernatants were collected to analyze the amount of total protein extracted using a bicinchoninic acid protein assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology). Protein lysates (20 µg) were subjected to 15 min of denaturation at 90˚C and 10% SDS-PAGE was performed before the proteins were transferred onto PVDF membranes (EMD Millipore). Subsequently, the membranes were subjected to 2 h blocking using 5% non-fat milk at 37˚C and incubated overnight at 4˚C with primary antibodies, including anti-NF-κB (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), anti-AQP4 (1:800; cat. no. ab46182; Abcam), anti-zonula occludens-1 (ZO-1; 1:400; cat. no. 61-7300; Invitrogen; Thermo Fisher Scientific, Inc.), anti-claudin-5 (1:1,000; cat. no. 35-2500; Invitrogen; Thermo Fisher Scientific, Inc.), anti-occludin (1:400; cat. no. 33-1500; Invitrogen; Thermo Fisher Scientific, Inc.), anti-GAPDH (1:1,000; cat. no. AF1186; Beyotime Institute of Biotechnology) and anti-Histone H3 (1:1,000; cat. no. AH433; Beyotime Institute of Biotechnology). Then, the membranes were rinsed with PBST containing 0.1% Tween-20 for 10 min and repeated five times. Subsequently, the PVDF membranes were incubated with a HRP-labeled goat anti-rabbit IgG (1:5,000; cat. no. abs20002; Absin Biotechnology Co., Ltd.) or HRP-labeled goat anti-mouse IgG secondary antibody (1:5,000; cat. no. abs20001; Absin) at 25˚C for 1 h. The bands were visualized using the ECL (cat. no. P0018; Beyotime Institute of Biotechnology) method and analyzed with the ImageJ software 1.8.0 (National Institutes of Health). GAPDH was used to normalize the expression of the proteins, whilst histone H3 was used to normalize the expression of NF-κB in the nuclear extracts.
Each value was measured three times and data are presented as the mean ± SD. SPSS 18.0 (IBM Corp.) was used for statistical analyses. No repeated-measure (matched) values were available in the current study, and all of the values were examined by one-way ANOVA and Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
MRI was first performed to determine the efficacy of HS treatment on lesions induced by TBI, as observed on the T2WI (
Compared with the sham group, the mNSS scores of the TBI group were found to be significantly increased at 48 h post-TBI. HS treatment significantly reduced the mNSS scores compared with those of TBI alone, suggesting that HS significantly reduced neurological deficits (P<0.05;
H&E staining results identified a regular arrangement of neurons in the sham group, along with capillary morphogenesis and normal glial cells. However, 2 days following TBI, the majority of cells were disorderly arranged where the nuclei became pyknotic or were severely shrunken. In addition, cellular swelling and hypervacuolization were observed in certain areas. HS treatment significantly improved TBI induced nuclei shrinking and cell swelling (
BBB leakage was next assessed by IHC staining for albumin and western blotting for claudin-5, ZO-1 and occludin. It was found that albumin staining was largely negative in the sham group, whilst a significant increase in albumin expression was observed in tissues from the TBI group (
In addition, tight junction (TJ)-associated proteins, including occludin, ZO-1 and claudin-5, were also revealed to be significantly downregulated by TBI, which was significantly reversed by HS treatment (
Astrocyte activation was evaluated by IHC staining for GFAP (
AQP4 expression tissues around the contusion site was evaluated using IHC (
AQP 4 expression in peri-TBI tissues was also measured using western blotting and RT-qPCR. Compared with the sham group, the protein and mRNA expression levels of AQP4 in the TBI group were found to be markedly increased, which was reversed by HS treatment.
The blood sodium concentration was analyzed using an automatic blood gas system, where the results showed that HS treatment remarkably increased the blood sodium ion concentration from 144.50±0.94 mmol/l (sham group) to 158.11±2.2 mmol/l (P<0.05;
The expression levels of inflammatory factors in each group were next measured (
Serum IL-1β levels were next measured using ELISA. Compared with the sham group, the serum IL-1β level in the TBI group was found to be significantly higher, which was significantly reversed by HS treatment. RT-qPCR was also performed in the tissue samples to detect the changes in IL-1β mRNA expression. Compared with the sham group, IL-1β mRNA expression in the TBI group was significantly higher, but HS treatment significantly downregulated IL-1β expression compared with that in the TBI group.
HS has previously demonstrated efficacy in treating brain edema in the clinic, but the precise underlying mechanism remains unknown. In the present study, MRI was performed to investigate the effects of HS on brain edema induced by TBI in rats. It was demonstrated that HS treatment significantly reduced the degree of brain edema in rats, restored the integrity of the BBB, reduced the activation of astrocytes and downregulated AQP4 expression. In addition, detection of inflammatory factors indicated that HS significantly reduced IL-1β and NF-κB expression.
AQP4 is the most important isoform of aquaporin in mammalian brain tissues, which regulate brain water homeostasis and has been previously demonstrated to be closely correlated with the formation of traumatic brain edema (
TBI-induced brain edema involves a highly complex mechanism, where the destruction of BBB integrity is an important link between vasogenic brain edema and secondary injuries (
The activation of astrocytes is mainly characterized by cell body hypertrophy of astrocytes with the notable thickening of protrusions (
Elevated inflammation is characteristic of brain edema, such that brain edema formation and posttraumatic inflammation are two key pathological processes that are associated with secondary brain injury (
NF-κB serves as a key player in the neuroinflammatory response in astrocytes during various neurological disorders by promoting the transcription of inflammatory mediators, including proinflammatory cytokines and chemokines (
However, some limitations associated with the current study should be noted. Firstly, the present study only evaluated brain edema at the end point of the experiments using T2WI. Therefore, monitoring of the progression of brain edema through different MRI sequences should be addressed in future studies. Additionally, although previous studies have reported cell death after TBI (
In conclusion, the present study identified the protective effect of tail vein injections of HS in a rat model of TBI-induced brain edema via MRI, BWC detection and histology. It was demonstrated that HS significantly reduced the upregulated expression of AQP4 induced by TBI. Further analysis of inflammatory factors suggested that the efficacy of HS in brain edema resulting from TBI was closely related to the downregulation of AQP4, the restoration of BBB integrity and the suppression of inflammatory factors IL-1β and NF-κB. However, the precise regulatory mechanism remains to be further elucidated.
Not applicable.
This work was supported by Projects of medical and health technology development program in Shandong province (grant no. 2017WS037).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HZ performed animal experiments and prepared this manuscript. JL performed the MRI experiment. YL performed the H&E and IHC experiments. CS measured the BWC. GF performed molecular biology experiments. WL performed statistical analysis. LF designed this study and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.
All of the animal procedures were conducted in accordance with the Guidelines for Care and Use of Laboratory Animals, and were approved by the Animal Care and Use Committee at Jining No. 1 People's Hospital (approval no. 2017037).
Not Applicable
The authors declare that they have no competing interests.
HS treatment reduces the TBI-induced lesion volume and cerebral swelling according to the T2WI. (A) White frame in the T2WI shows the brain region examined in the current study. (B) Representative T2WI of rats in the sham, vehicle-treated and HS-treated rat groups captured 2 days after TBI. (C) Quantitative analysis of the corrected lesion volume using the T2WI of each group 2 days after TBI. (D) Quantitative analysis of brain swelling using the T2WI 2 days following TBI. Data are presented as the mean ± SD. ***P<0.001 vs. Sham group; #P<0.05 vs. TBI group. TBI, traumatic brain injury; T2WI, T2-weighted image; HS, hypertonic saline.
HS improves TBI-induced neurological functional deficits and brain edema. (A) HS treatment significantly reduced neurological deficits at 48 h post-TBI. (B) Brain water content was measured in each rat from the sham, TBI and TBI + HS treatment groups to evaluate brain edema. (C) Representative hematoxylin and eosin staining images identified that TBI-induced pyknotic and shrunken nuclei, and cellular edema, which were ameliorated by HS treatment. White arrows indicate the injured cells with pyknotic or shrunken nuclei induced by cellular edema. Magnification, x400. Data are expressed as the mean ± SD. #P<0.05 vs. TBI group. **P<0.01 vs. the sham group. TBI, traumatic brain injury; HS, hypertonic saline; mNss, modified neurological severity score.
HS improves TBI-induced BBB injury. (A) BBB integrity was evaluated by immunohistochemical staining for albumin. Magnification, x400. (B) Average optical density of albumin staining from the three treatment groups. (C) Expression levels of occludin, ZO-1 and claudin-5 were evaluated by western blotting and (D) Quantified. Data are presented as the mean ± SD. #P<0.05 and ##P<0.01 vs. TBI group. **P<0.01, ***P<0.001 vs. the sham group. TBI, traumatic brain injury; HS, hypertonic saline; BBB, blood brain barrier; ZO-1, zonula occludens-1.
HS reduces TBI-induced astrocyte activation. (A) Astrocyte activation was evaluated by immunohistochemical staining for GFAP. Magnification, x400. TBI increased the expression of GFAP along with astrocyte hyperplasia, cell body enlargement, protrusion and thickening, as well as extravascular collagen fibers, which were ameliorated by HS treatment. White arrows indicate the thickening extravascular collagen fibers. (B) Average optical density of GFAP staining. Data are presented as the mean ± SD. ##P<0.01 vs. TBI group. ***P<0.001 vs. sham group. TBI, traumatic brain injury; HS, hypertonic saline; GFAP, glial fibrillary acidic protein.
HS treatment reduces AQP4 expression in cerebral cortical tissues around the contusion site. (A) Representative images of immunohistochemical staining for AQP4 indicated that TBI-induced AQP4 upregulation was ameliorated by HS treatment. White arrows indicate the AQP4 positive staining around the blood vessels. Magnification, x400. (B) Average optical density of AQP4 staining. (C) Western blotting and (D) reverse transcription-quantitative PCR were utilized to detect the mRNA and protein expression levels of AQP4 in each group 2 days after TBI. Data are presented as the mean ± SD. #P<0.05, ##P<0.01 vs. TBI group. ***P<0.001 vs. the sham group. TBI, traumatic brain injury; HS, hypertonic saline; AQP4, aquaporin 4.
HS treatment reduces TBI-induced inflammation. (A) NF-κB protein expression in cerebral cortical tissues around the contusion site was determined by western blotting. (B) Quantitative analysis of NF-κB mRNA and protein expression levels in each group. (C) Plasma IL-1β concentrations and the tissue mRNA expression of IL-1β were evaluated by ELISA and reverse transcription-quantitative PCR, respectively. Data are presented as the mean ± SD. #P<0.05 vs. TBI group. ***P<0.001 vs. the sham group. TBI, traumatic brain injury; HS, hypertonic saline; IL, interleukin.
Blood sodium concentrations in each treatment group.
Treatment group | Sodium concentration, mmol/l |
---|---|
Sham | 144.50±0.94 |
TBI | 149.75±1.33 |
TBI + HS | 158.11±2.2 |
aP<0.05 vs. Sham. TBI, traumatic brain injury; HS, hypertonic saline.