Background
Spinal cord injury (SCI) is responsible for heavy societal and economic burdens [
1]. Currently, SCI can only be relieved by surgery, and cannot be cured [
2]. Endothelial cells (ECs), the basement membrane, pericytes and the terminal foot processes of astrocytes constitute the blood-spinal cord barrier (BSCB), which normally protects the parenchyma [
3,
4]. After SCI, barrier integrity is compromised by the disruption of interendothelial tight junctions (TJs) and adherens junctions (AJs) as well as overall mechanical damage to vessels. This compromise in the BSCB results in the infiltration of immune cells and neurotoxic products, causing the death of nerve cells, and permanent neurological dysfunction [
5‐
7]. Therefore, there is a crucial need to identify interventions that can effectively prevent BSCB destruction after SCI.
Matrix metalloproteinases (MMPs), a family of zinc-containing peptidases that degrade and reshape the extracellular matrix and other extracellular proteins, play a key role in barrier function [
7,
8]. Studies have shown that MMPs exacerbate the destruction of the BBB/BSCB under pathological conditions, including SCI [
8,
9]. Two important members of the MMP superfamily are MMP-2 and -9 [
10]. MMP-9 can induce BSCB-related protein degradation, and the upregulation of MMP-2 can lead to initial opening of the BBB/BSCB [
11,
12]; in contrast, blocking MMP-9 activity can protect against vascular permeability [
9]. MMP-2/9 expression has been detected up to 7 days after SCI [
13]. Vascular endothelial growth factor (VEGF) is a highly specific vascular EC growth factor that promotes vascularisation, proliferation, and angiogenesis [
14]. Additionally, VEGF is a necessary angiogenic factor for embryonic development and neovascularization under many pathological conditions [
15]. Several molecules have been demonstrated to be angiogenic in vivo, but only VEGF is considered to be a secretory mitogen specific to vascular ECs. At present, VEGF is the most likely target for the study of vascular growth dynamics [
16,
17].
Previous studies on this topic have mainly focused on drug treatment after SCI [
18,
19]. However, these treatments often have specific side effects, affecting patient quality of life. Therefore, it is important to identify a safe, effective and healthy treatment for patients with SCI. Exercise training is a non-traumatic rehabilitation method that can promote the functional recovery of paralyzed muscles [
20‐
23]. To date, most studies have focused on the effects of drug therapy on the neurovascular system after SCI [
24,
25], but ignored the role of exercise training in protection and functional recovery of the vascular system. Based on this fact and starting from clinical practice to simulate exercise rehabilitation in patients, our experimental team has designed and invented the first water treadmill that is suitable for rats to exercise after SCI. Compared with swimming exercise, water treadmill training(TT) causes rats to passively and forcibly exercise at the initial stage, after which rats actively follow the treadmill in the middle and later stages of training. It is difficult for rats to perform rehabilitation exercise on an ordinary treadmill due to its high resistance. Our water treadmill equipment combines swimming and TT, which addresses the above shortcomings. The protective effects of TT on SCI have not been reported in the literature, and its effects on the BSCB are unclear. The aim of these experiments was to measure the protective effects of TT on the BSCB after SCI and investigate the mechanisms involved.
Evaluation of BSCB permeability
Water content
At 7 or 14 d after SCI, 2% sodium pentobarbital was intraperitoneally injected to anaesthetize the animals (
n = 5), after which the rats underwent cardiac perfusion with 0.9% normal saline and 0.5 cm of the T10 spinal cord segment was removed. The degree of oedema in this segment was assessed by the dry and wet weight method as previously reported [
28,
29].
Evans blue (EB) dye assay
According to previously reported methods [
4,
8], rats (
n = 5) were injected with EB dye (4 ml/kg) by the tail vein at 7 or 14 d after SCI to assess BSCB permeability, followed by treatment with 2% sodium pentobarbital anaesthesia 2 h later and 0.9% saline perfusion. Tissues containing T10 were soaked in N,N′-dimethylformamide at 50 °C for 72 h. The concentration of EB dye in the samples was determined based on a standard curve (μg/g). Tissues were cut into 15-μm thick sections with a freezing microtome at − 20 °C, and then analysed. Quantitative data analysis was performed with ImageJ software.
Haematoxylin–eosin (HE) staining
Briefly, T9–T11 spinal cord tissue was removed from the rats at 7 or 14 d after SCI, and stored in 4% paraformaldehyde for 24 h (4 °C). The spinal cord tissue was immersed in a 0.1 M phosphate buffer solution and 30% sucrose solution overnight (4 °C). Successive sections (15-μm thick) were frozen and stored for subsequent HE staining.
Western blot analysis
Tissues containing T10 segments were put into a collection tube containing a mixture of phenylmethanesulfonyl fluoride (PMSF) and RIPA lysis buffer (100:1) and then microcentrifuged at 12,000 rpm for 5 min at 4 °C. We extracted the supernatant and calculated the protein concentration with a BCA protein assay kit. The mixed solution was heated to 100 °C for 10 min. After electrophoretic transfer to membranes, they were incubated with the appropriate primary (anti-p120-Catenin, anti-β-Catenin, anti-ZO-1, anti-Occludin, anti-Claudin-5, anti-MMP-9, anti-MMP-2, anti-VEGF, anti-Tubulin, or anti-β-Actin) and secondary antibodies, and the signal was digitally quantified.
Immunofluorescence staining
After the sections had been dried, they were washed 3 times for 15 min. They were treated with non-immune goat serum for 1 h and then incubated with the following primary antibodies for 50 min at room temperature: rabbit anti-Occludin antibody (1:100), rabbit anti-claudin-5 antibody (1:100), rabbit anti-p120-Catenin (1:200), rabbit anti-β-Catenin (1:100), anti-CD31(1:100), anti-BrdU (1:100) and anti-Laminin (1:100) at 4 °C. This was followed by incubation with Alexa Fluor 488 Affinipure goat anti-rabbit IgG (H + L) (1:200, Yeasen, China) for 50 min at room temperature. Phosphate-buffered saline (PBS) was used in place of the primary antibody as a negative control. We use an in situ cell death detection kit to detect apoptotic cells. The nuclei were coloured by staining with Hoechst or DAPI. The fluorescence signal was observed by laser confocal microscopy. Five fields on each of three slides per animal were randomly selected for visualization and analysis performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Transmission electron microscopy (TEM)
A total of 18 mice were subjected to TEM. Tissue was quickly removed, cut into 1 mm3 pieces on ice and soaked in 2.5% glutaraldehyde. The tissue was then fixed with a 1% oxidizing fixative for 1 h, stained with 1% uranyl acetate for 2 h, and embedded after dehydration in a gradient acetone solution. After semi-thin sectioning and toluidine blue staining, ultrathin sections were cut and observed using a Hitachi transmission electron microscope.
Images were taken under the same conditions, including brightness and contrast, to better compare TJs among different groups. According to standard protocols [
30,
31], the width and length of the TJs were blindly measured and averaged by two examiners using ImageJ software.
Statistical analyses
All experimental data are expressed as the mean ± standard deviation. The Kolmogorov–Smirnov (K–S) test was used as a normality test, with p > 0.05 indicating a normal distribution, Levene’s test was used as a test of homogeneity of variance, with p > 0.05 used to indicate homogeneous variance, and vice versa. A t-test was used to compare two groups. One-way ANOVA and Dunnett’s test were used to evaluate the data when more than two groups were compared. Statistical analyses were performed with SPSS 16 statistical software, and p < 0.05 was used to indicate statistical significance.
Discussion
Our data in this study indicate that TT can protect the integrity of the BSCB and prevent further spinal cord oedema after impact injury. After TT, the histological structure and motor function of the spinal cord were significantly improved. We showed that TT could promote angiogenesis, reduce apoptosis and inhibit MMP-2/9 expression. Therefore, the vascular protection of the BSCB by TT may occur through the following mechanisms: TT (1) protects the residual BSCB structure from further damage, (2) promotes vascular regeneration, and (3) inhibits MMP-2/9 expression to mitigate BSCB damage.
Emerging evidence indicates that exercise therapy can promote recovery after SCI [
32‐
35]. It is well known that normal exercise training cannot be carried out by rats after SCI. Thus, we introduced water TT, which simulates clinical exercise treatment and can help rats successfully train on a treadmill in the early stage of SCI. TT for human patients is mainly used in rehabilitation for motor system diseases [
36,
37]. TT can reduce the resistance of forward movement, allowing patients to walk or run with a normal gait [
38]. Additionally, the depth and speed of TT can be adjusted, which facilitates the control of exercise intensity [
38].
For the first time, mechanisms to mitigate damage after SCI were studied through our experiments in rats. Trauma, infection, tumour growth or obstruction of the blood supply can cause oedema of the central nervous system (CNS). Cytotoxicity and vasogenic edema are interdependent factors involved in the development of CNS oedema. Prolongation of cytotoxic oedema leads to vasogenic oedema, and vice versa [
39,
40]. Cytotoxic oedema, which refers to the accumulation of water in intact cells, occurs when an anoxic state leads to the loss of energy-dependent solute homeostasis [
41]. Vasogenic oedema refers to the accumulation of fluid in the extracellular space around the damaged BBB/BSCB [
42]. The integrity of the BSCB is essential for the spinal cord to maintain its normal function [
43]. Destruction of the BSCB after SCI, leads to increased permeability, causing secondary damage [
18,
44]. In our study, through the horizontal comparison of data from each group collected at the same time point, we found that the degree of oedema in the SCI group was increased significantly higher than that in the sham-operated group; this was accompanied by lower BBB motor scores, severe tissue structure damage and a large number of necrotic cells. However, these conditions were significantly improved after TT, which may be related to the protective effect of TT on the BSCB. Longitudinal comparison of the BBB scores 7 and 14 d after SCI showed some recovery with time (Fig.
2c). The partial recovery of these functions may be related to both the preservation of BSCB function and formation of new blood vessels.
The BSCB protects the spinal cord by restricting the entry of plasma components and blood cells [
9]. Following SCI, destruction of the BSCB leads to increased microvascular permeability, inflammatory reactions, tissue oedema, and neurotoxic products [
45]. TJs, AJs, and gap junctions connect the ECs lining microvessels in the spinal cord [
3,
46]. The dense network of TJs and AJs is destroyed after SCI, resulting in the decreased expression of TJ and AJ proteins. Our results show thatp120-Catenin, β-Catenin, ZO-1, Occludin, and Claudin-5 expression was greatly reduced after SCI. However, their expression was significantly improved in the TT-treated spinal cord (Figs.
3,
4). This shows that the residual BSCB structure had been protected by TT.
To determine the protective mechanism of TT, we performed more in-depth research. VEGF stimulates EC proliferation and survival vascular structure formation, nitric oxide-dependent vasodilatation and vascular leakage [
17]. It has been reported that VEGF peaks at 3 d, and a large amount of VEGF remained at 7 d after SCI [
47]. In addition, ischaemia and injury can induce angiogenesis, which provide oxygen and nutrition to the ischaemic or diseased site, thus improving tissue repair and remodeling [
48‐
50]. We used laminin as a vascular marker and BrdU as a proliferation marker [
51]. The results showed that TT could promote angiogenesis after SCI, as shown by the detection of VEGF protein expression. Quantitative analysis of neovascularization 7 d after SCI with co-labelling also showed that the number of BrdU
+/Laminin
+ cells increased significantly after TT treatment in rats with SCI, helping to maintain the stability of the BSCB (Fig.
5).
The activation of MMP-2/9 after SCI plays an essential role in the destruction of the BBB/BSCB [
9,
10], and MMP-2/9 expression can aggravate damage [
52]. In this study, 7 d after SCI, MMP-2/9 expression was upregulated (Fig.
6a–c), showing their potential to further aggravate and damage the BSCB. We were surprised to find that MMP-2/9 expression was significantly decreased after TT. Although we have not proven how TT downregulates MMP-2/9 expression, we believe that TT can effectively prevent the destruction of the BSCB, partially through the inhibition of MMP-2/9 expression after SCI.
As far as we know, this is the first time that TT has been applied in the treatment of SCI in rats. We found that TT could enhance the expression of TJ and AJ proteins after SCI. We also found that TT could promote angiogenesis and inhibit MMP-2/9 expression, which may be an important mechanism by which TT maintains the stability of the BSCB. These experimental results provide a better understanding of the possible mechanisms of TT in the treatment of SCI and a reliable basis for the application of TT in the future.
We acknowledge the limitations of our experiments. In this study, we only used male rats, ignoring any possible differences caused by sex. Due to limitations in the experimental conditions, we could not observe changes at the functional level through electrophysiological techniques to understand the role of TT. The training period was relatively short and the specific mechanism by which TT inhibits MMP-2/9 expression remains to be further studied.
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